Transfection, storage and transfer of male germ cells for generation of transgenic species &amp; genetic therapies

ABSTRACT

Disclosed is an in vivo method of incorporating exogenous genetic material into the genome of a vertebrate, which involves administering to a male vertebrate&#39;s testis a gene delivery mixture comprising a viral vector, such as a retroviral vector, to deliver a polynucleotide encoding a desired trait or product. Also disclosed is an in vitro method of incorporating exogenous genetic material into the genome of a vertebrate, in which germ cells are obtained from a donor male vertebrate and are genetically modified in vitro, before being transferred to a recipient male vertebrate. After the transfer, the male vertebrate bearing the genetically modified germ cells is bred with a female vertebrate such that a transgenic progeny is produced that carries the polynucleotide in its genome. Also disclosed are non-human transgenic vertebrates produced in accordance with the method, including transgenic progeny. A transgenic cell derived from the transgenic vertebrate is also disclosed, being a germ cell, such as a spermatozoan or ovum, a precursor cell of either of these, or a somatic cell. A method of producing a non-human transgenic vertebrate animal line comprising native germ cells carrying in their genome at least one xenogeneic polynucleotide is disclosed, as is vertebrate semen containing the transgenic male germ cells useful in practicing the method.

This application is a continuation-in-part of U.S. Ser. No. 09/311,599,filed May 13, 1999, which is a continuation-in-part of U.S. Ser. No.09/191,920, filed Nov. 13, 1998. This application claims the benefit ofU.S. Provisional Application No. 60/065,825 which was filed on Nov. 14,1997.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

1.The Field of the Invention

This invention relates to the medical arts, particularly to the field oftransgenics and gene therapy. The invention is particularly directed toin vitro and in vivo methods for genetically modifying male germ cellsand support cells (i.e., Leydig and Sertoli cells), which methodsincorporate a method depopulating a vertebrate testis of male germcells.

2. Discussion of Related Art

The field of transgenics was initially developed to understand theaction of a single gene in the context of the whole animal and thephenomena of gene activation, expression, and interaction. Thistechnology has been used to produce models for various diseases inhumans and other animals. Transgenic technology is amongst the mostpowerful tools available for the study of genetics, and theunderstanding of genetic mechanisms and function.

It is also used to study the relationship between genes and diseases.About 5,000 diseases are caused by a single genetic defect. Morecommonly, other diseases are the result of complex interactions betweenone or more genes and environmental agents, such as viruses orcarcinogens. The understanding of such interactions is of primeimportance for the development of therapies, such as gene therapy anddrug therapies, and also treatments such as organ transplantation. Suchtreatments compensate for functional deficiencies and/or may eliminateundesirable functions expressed in an organism.

Transgenesis has also been used for the improvement of livestock, andfor the large scale production of biologically active pharmaceuticals.Historically, transgenic animals have been produced almost exclusivelyby microinjection of the fertilized egg. The pronuclei of fertilizedeggs are microinjected in vitro with foreign, i.e., xenogeneic orallogeneic DNA or hybrid DNA molecules. The microinjected fertilizedeggs are then transferred to the genital tract of a pseudopregnantfemale. (E.g., P. J. A. Krimpenfort et al., Transgenic mice depleted inmature T-cells and methods for making transgenic mice, U.S. Pat. Nos.5,175,384 and 5,434,340; P. J. A. Krimpenfort et al., Transgenic micedepleted in mature lymphocytic cell-type, U.S. Pat. No. 5,591,669).

This widely used technique requires large numbers of fertilized eggs,equipment to handle embryos and the facility to microinject them invitro. This is partly because there is a high rate of egg loss due tolysis during microinjection. Moreover manipulated embryos are lesslikely to implant and survive in utero. These factors contribute to thetechnique's extremely low efficiency. Superovulated mammals (e.g.,primates, cows, horses, pigs, and mice) produce only 10-20 or less eggsper female animal per cycle, even after hormonal stimulation, and only1% of microinjected mouse eggs (Palmiter, R. D. and Brinster, R. L.,Germline transformation of mice, Annu. Rev. Genet. 20:465-99 [1986]),and 0.1% of cattle, sheep and pig eggs (Wall, R. J., et al., Makingtransgenic livestock: genetic engineering on a large scale, J. CellBiochem. 49:113-120 [1992]) develop into transgenic animals. Typically,300-500 fertilized eggs must be microinjected to produce perhaps threetransgenic animals. Consequently, generating large animals with thesetechniques is prohibitively expensive. For this reason, mammaliantransgenic technology has been confined almost exclusively to mice dueto their high fecundity. Little has been done to improve the generationof transgenic animals by the microinjection of a transgene intofertilized eggs (Gordon, J. and Ruddle, F. H., Integration and stablegerm line transmission of genes injected into mouse pronuclei, Science214:1244-1246 [1981]).

While small animals such as mice have proved to be suitable models forcertain diseases, their value in this respect is limited. Largertransgenic animals would be much more suitable than mice for the studyof the effects and treatment of most human diseases because of theirgreater similarity to humans in many aspects, and better for studyingorgan systems or behavior. Larger mammals are also more suitable thanmice as potential organ donors to humans due to the comparable size oftheir organs. Now that transgenic animals with the potential for humanxenotransplantation are being developed, more of these larger animalswill be required. Transgenic technology will allow that such donoranimals will be immunocompatible with the human recipient.

In contrast to only 10-20 eggs per female even after treatment withsuperovulatory drugs, most male mammals, including mice and nearly alllarger mammals, generally produce at least about 10⁸ spermatozoa (malegerm cells) in each ejaculate. For this reason alone, male germ cellswill be a better target for introducing foreign DNA into the germ line,leading to the generation of transgenic animals with increasedefficiency and after simple, natural mating.

Nevertheless, attempts to generate transgenic mice using spermatozoa tocarry DNA into the egg (Lavitrano, M., et al., Sperm cells as vectorsfor introduction of DNA into eggs: genetic transformation of mice, Cell57: 717-723 [1989]; WO-A-90/08192), have not been validated (Brinster,R. L., et al., No simple solution for making transgenics, Cell59:239-241 [1989]). Recently, transgenic mice were produced after theinjection of exogenous DNA together with sperm heads into oocytes (Perry, A. C., et al., Mammalian transgenesis by intracytoplasmic sperminjection, Science 284:1180-1183 [1999]). Following uterine transfer,20% of these embryos developed into transgenic offspring.

Genetic information has been transferred to embryos using retroviralvectors (Jaenisch, R., Germ line integration and Mendelian transmissionof the exogenous Moloney leukemia virus, Proc. Natl. Acad. Sci. USA73:1260-1264 [1976]), but the animals were mosaics with different geneinsertions in different tissues. (Jaenisch, R., Retroviruses andembryogenesis: microinjection of Moloney leukemia virus intomidgestation mouse embryos, Cell 19:181-188 [1980]). Recently, fivetransgenic calves were produced by injection of a pseudotypedreplication-deficient vector based on the Moloney murine leukemia virus.The vector was introduced into the perivitelline space of metaphase IIoocytes (Chan, A. W., et al., Transgenic cattle produced byreverse-transcribed gene transfer in oocytes, Proc. Natl. Acad. Sci. USA95:14028-14033 [1998]).

An alternative, not yet fully realized, is the stable transfection ofmale germ cells in vitro and their transfer to a recipient testis.Transfer of genetically marked germ cells to the testis yieldedoffspring, but so far no transgenic progeny have been produced(Brinster, R. L. and Avarbok, M. R., Germline transmission of donorhaplotype following spermatogonial transplantation, Proc. Natl. Acad.Sci. USA 91:11303-11307 [1994]).

Spermatogenesis is the process by which a diploid spermatogonial stemcell provides daughter cells which undergo dramatic and distinctmorphological changes to become self-propelling haploid cells (malegametes) capable, when fully mature, of fertilizing an ovum.

Primordial germ cells are first seen in the endodermal yolk sacepithelium at E8 and are thought to arise from the embryonic ectoderm(A. McLaren and M. Buehr, Cell Diff. Dev. 31:185 [1992]; Y. Matsui etal., Nature 353:750 [1991]). They migrate from the yolk sac epitheliumthrough the hindgut endoderm to the genital ridges and proliferatethrough mitotic division to populate the testis.

At sexual maturity the spermatogoniium goes through 5 or 6 mitoticdivisions before it enters meiosis. The primitive spermatogonial stemcells (A0/As) proliferate and form a population of intermediatespermatogonia types Apr, Aal, A1-4 after which they differentiate intotype B spermatogonia. The type B spermatogonia differentiate to formprimary spermatocytes which enter a prolonged meiotic prophase duringwhich homologous chromosomes pair and recombine. The states of meiosisthat are morphologically distinguishable are; preleptotene, leptotene,zygotene, and pachytene; secondary spermatocytes and the haploidspermatids are later stages. Spermatids undergo great morphologicalchanges during spermatogenesis, such as reshaping the nucleus, formationof the acrosome and assembly of the tail (A. R. Bellve et al., Recovery,capacitation, acrosome reaction, and fractionation of sperm, MethodsEnzymol. 225:113-36 [1993]). The spermatocytes and spermatids establishvital contacts with the Sertoli cells through unique hemi-junctionalattachments with the Sertoli cell membrane. The final changes in thematuring spermatozoan (i.e., spermatozoon) take place in the genitaltract of the female prior to fertilization.

Initially, attempts were made to produce transgenic animals by addingDNA to spermatozoa which were then used to fertilize mouse eggs invitro. The fertilized eggs were then transferred to pseudopregnantfoster females, and of the pups born, 30% were reported to be transgenicand express the transgene. Despite repeated efforts by others, however,this experiment could not be reproduced and no transgenic pups wereobtained. Indeed, there remains little doubt that the transgenic animalsreputed to have been obtained by this method were not transgenic at alland the DNA incorporation reported was mere experimental artifact. Datacollected from laboratories around the world engaged in testing thismethod showed that no transgenics were obtained from a total of 890 pupsgenerated.

In summary, it is currently possible to produce live transgenic progenybut the j available methods are costly and extremely inefficient.Spermatogenic transfection in accordance with this invention, either invitro or in vivo, provides a simple, less costly and less invasivemethod of producing transgenic animals and one that is potentiallyhighly effective in transferring allogeneic as well as xenogeneic genesinto the animal's germ cells.

To facilitate in vitro transfection of male germ cells and implantationinto a-testis of a recipient male vertebrate it is advantageous first todepopulate the testis of the recipient vertebrate of untransfected malegerm cells before transferring transfected male germ cells into it.

Depopulation of testis has commonly been done by exposing the wholevertebrate to gamma irradiation (X-ray), or localizing irradiation tothe testis. (E.g., G. Pinon-Lataillade et al., Endocrinological andhistological changes induced by continuous low dose gamma-irradiation ofrat testis, Acta Endocrinol. (Copenh) 109(4):558-62 [1985]; G.Pinon-Lataillade and J. Maas, Continuous gamma-irradiation of rats:dose-rate effect on loss and recovery of spermatogenesis,Strahlentherapie 161(7):421-26 [1985]; C. R. Hopkinson et al., Theeffect of local testicular irradiation on testicular histology andplasma hormone levels in the male rat, Acta Endocrinol. (Copenh)87(2):413-23 [1978]; G. Pinon-Lataillade et al., Influence of germ cellsupon Sertoli cells during continuous low-dose rate gamma-irradiation ofadult rats, Mol. Cell Endocrinol. 58(1):51-63 [1988]; P. Kamtchouing etal., Effect of continuous low-dose rate gamma-irradiation on rat Sertolicell function, Reprod. Nutr. Dev. 28(4B):1009-17 [1988]; C. Pineau etal., Assessment of testicularfunction after acute and chronicirradiation: further evidence for influence of late spermatids onSertoli cell function in the adult rat, Endocrinol. 124(6):2720-28[1989]; M. Kangasniemi et al., Cellular regulation of basal andFSH-stimulated cyclic AMP production in irradiated rat testes, Anat.Rec. 227(1):32-36 [1990]; G. Pinon-Lataillade et al., Effect of an acuteexposure of rat testes to gamma rays on germ cells and on Sertoli andLeydig cell functions, Reprod. Nutr. Dev. 31(6):617-29 [1991]).

The mechanism of gamma radiation-induced spermatogonial degeneration isthought to be related to the process of apoptosis. (M. Hasegawa et al.,Resistance of differentiating spermatogonia to radiation-inducedapoptosis and loss in p53-deficient mice, Radiat. Res. 149:263-70[1998]).

Another method of depopulating a vertebrate testis is by administering acomposition containing an alkylating agent, such as busulfan (Myleran).(E.g., F. X. Jiang, Behaviour of spermatogonia following recovery frombusulfan treatment in the rat, Anat. Embryol. 198(1):53-61 [1998]; L. D.Russell and R. L. Brinster, Ultrastructural observations ofspermatogenesis following transplantation of rat testis cells into mouseseminiferous tubules, J. Androl. 17(6):615-27 [1996]; N. Boujrad et al.,Evolution of somatic and germ cell populations after busulfan treatmentin utero or neonatal cryptochidism in the rat, Andrologia 27(4):223-28[1995]; R. E. Linder et al., Endpoint of spermatotoxicity in the ratafter short duration exposures to fourteen reproductive toxicants,Reprod. Toxicol. 6(6):491-505 [1992]; F. Kasuga and M. Takahashi, Theendocrine function of rat gonads with reduced number of germ cellsfollowing busulfan treatment, Endocrinol. Jpn 33(1):105-15 [1986]).

Cytotoxic alkylating agents, such as busulfan, chlorambucil,cyclophospharnide, melphalan, or ethyl ethanesulfonic acid, arefrequently used to kill malignant cells in cancer chemotherapy. (E.g.,Andersson et al., Parenteral busulfan for treatment of malignantdisease, U.S. Pat. Nos. 5,559,148 and 5,430,057; Stratford et al.,Stimulation of stem cell growth by the bryostatins, U.S. Pat. No.5,358,711; Luck et al., Treatment employing vasoconstrictive substancesin combination with cytotoxic agents for introduction into cellularlesion, U.S. Pat. No. 4,978,332). Treatment of mice with busulfan (13mg-40 mg/kg body wt.), was reported to deplete male germs cells in thetestis; both stems cells and differentiating spermatogonia were killed;doses over 30 mg/kg body weight resulted in azoospermia for up to 56days after treatment. (L. R. Bucci and M. L. Meistrich, Effects ofbusulfan on murine sperrmatogenesis: cytotoxicity, sterility, spermabnormalities and dominant lethal mutations, Radiation Research176:259-68 [1987]).

The present invention addresses the need for spermatogenic geneticmodification, either in vitro or in vivo, that is highly effective intransferring allogeneic as well as xenogeneic genes into the animal'sgerm cells and in producing transgenic vertebrate animals. The presenttechnology addresses the requirements of germ line and stem cell linegene therapies in humans and other vertebrate species, including theneed for a superior method of depopulating a testis of untransfectedmale germ cells. The present technology is of great value in producingtransgenic animals in large species as well as for repairing geneticdefects that lead to male infertility. Male germ cells that have stablyintegrated the DNA are selectable. These and other benefits and featuresof the present invention are described herein.

SUMMARY OF THE INVENTION

The present invention arose from a desire by the present inventors toimprove on existing methods for the genetic modification of an animal'sgerm cells and for producing transgenic animals. The pre-existing artmethods rely on direct injection of DNA into zygotes produced in vitroor in vivo, or by the production of chimeric embryos using embryonalstem cells incorporated into a recipient blastocyst. Following this,such treated embryos are transferred to the primed uterus or oviduct.These prior methods are extremely slow and costly, rely on severalinvasive steps, and only produce transgenic progeny sporadically andunpredictably.

In their search for a less costly, faster, and more efficient approachfor producing transgenics, the present inventors devised the presentmethod which relies on the in vivo or in vitro (ex vivo) geneticmodification of vertebrate male germ cells with a nucleic acid segment,i.e., a polynucleotide, encoding a desired trait or product.

The present invention relates to the in vivo and in vitro (ex vivo)genetic modification, for example, by transfection or transduction, ofvertebrate animal germ cells with a desired genetic material. Briefly,the in vivo method involves injection of genetic material together witha suitable vector directly into the testicle of the animal. In thismethod, all or some of the male germ cells within the-testicle aregenetically Emodified in situ, under effective conditions. The in vitromethod involves obtaining germ cells from the gonad (i.e., testis) of asuitable donor or from the animal's own testis, using a novel isolationor selection method, transfecting or otherwise genetically altering themin vitro, and then returning them to the substantially depopulatedtestis of the donor or of a different recipient male vertebrate undersuitable conditions where they will spontaneously repopulate thedepopulated testis. The in vitro method has the advantage that thetransfected germ cells can be screened by various means before beingreturned to the testis of the same or a different suitable recipientmale to ensure that the transgene is incorporated into the genome in astable state. Moreover, after screening and cell sorting only enrichedpopulations of germ cells can be returned. This approach provides agreater chance of transgenic progeny after mating.

In particular, the inventive in vivo method of incorporating exogenousgenetic material into the genome of a vertebrate involves administeringto a male vertebrate's testis(es) a gene delivery mixture comprising aviral vector, such as, but not limited to, a retroviral vector, thatcomprises at least one polynucleotide defining a gene encoding a desiredtrait or product and, optionally, a gene encoding a genetic selectionmarker. The gene(s) are operatively linked to a promoter sequence (allthe individual genes used are not necessarily linked to a singlepromoter sequence), such that a transcriptional unit is formed, and areadministered under conditions effective to reach at least one of thespermatozoa, or precursors of spermatozoa, residing in the vertebrate'stestis. The delivery mixture, including the polynucleotide(s), areadministered in amounts and under conditions effective such that apolynucleotide encoding a desired trait or product is incorporated intothe genome of at least one male germ cell, such as a spermatozoan orprecursor cell, so that a genetically modified male gamete is producedby the male vertebrate. Then, the male vertebrate is bred, naturally orwith the aid of artificial reproductive technologies, with a femalevertebrate of its species such that a transgenic progeny is therebyproduced that carries the polynucleotide in its genome.

The invention also includes an in vitro method of incorporating at leastone polynucleotide encoding a desired trait or product into the genomeof a vertebrate. The F in vitro method:involves obtaining from a donormale vertebrate a male germ cell, such as a spermatozoan cell or aprecursor cell, and genetically modifying the cell in vitro with atleast one polynucleotide encoding a desired trait or product other thanan immortalizing molecule, and a polynucleotide defining a gene encodinga genetic selection marker, in the presence of a gene delivery mixturecomprising a viral vector, at about or below the vertebrate's bodytemperature and for an effective period of time such that thepolynucleotide encoding a desired trait or product is incorporated intothe genome of the cell. Then the genetically modified germ cell isisolated or selected, with the aid of the genetic selection markerexpressed in the genetically modified cell, and transferred to a testisof a recipient male vertebrate such that the cell lodges in aseminiferous tubule of the testis, such that a genetically modified malegamete is produced therein. The male vertebrate is bred with a femalevertebrate of its species such that a transgenic progeny is therebyproduced that carries the polynucleotide in its genome.

This invention also relates to a non-human transgenic male vertebrateproduced in accordance with either the in vivo or in vitro method ofincorporating exogenous genetic material into the genome of avertebrate. Produced in accordance with the in vivo method, thetransgenic vertebrate is the recipient of the gene delivery mixture.Produced in accordance with the in vitro method, the transgenicvertebrate is the recipient of the genetically modified male germ cellthat was transferred to its testis. The transgenic male vertebrate canbe bred with a female of its species, because it comprises a native malegerm cell carrying in its genome a polynucleotide of exogenous origindefining a gene encoding a desired trait or product. But somatic cellsin tissues outside the testis of the transgenic vertebrate lack thepolynucleotide.

This invention also relates to a non-human transgenic vertebrateproduced in accordance with either the in vivo or in vitro method ofincorporating exogenous genetic material into the genome of avertebrate. The non-human transgenic vertebrate is the direct orindirect progeny of the male vertebrate that received the gene deliverymixture, in accordance with the in vivo method. Alternatively, thenon-human transgenic vertebrate is the direct or indirect progeny of therecipient of the genetically modified male germ cell that wastransferred to its testis, in accordance with the in vitro method. Thus,the transgenic progeny is the immediate offspring of the transgenic malevertebrate, or is an offspring thereof separated by one or moregenerations. The transgenic vertebrate includes one or more cellscarrying in their genome a polynucleotide of exogenous origin thatencodes a desired trait or product.

Also, the invention includes a transgenic cell derived from thetransgenic progeny. The cell is a germ cell, such as a spermatozoan(i.e., spermatozoon) or ovum, a precursor cell of either of these, or asomatic cell.

The invention also relates to vertebrate semen containing a plurality ofthe inventive transgenic male germ cell.

The invention is also directed to a method of producing a non-humantransgenic vertebrate animal line comprising native germ cells carryingin their genome at least one xenogeneic polynucleotide. The methodinvolves breeding of the transgenic progeny with a member of theopposite sex of the same species; and selecting its progeny for thepresence of the polynucleotide.

This technology is applicable to the production of transgenic animalsfor use as animal models, and to the modification of the genome of ananimal, including a human, by addition, modification, or subtraction ofgenetic material, often resulting in phenotypic changes. The presentmethods are also applicable to altering the carrier status of an animal,including a human, where that individual is carrying a gene for arecessive or dominant gene disorder, or where the individual is prone topass a multigenic disorder to his offspring.

These and other advantages and features of the present invention will bedescribed more fully in a detailed description of the preferredembodiments which follows. In further describing the invention, thedisclosures of related applications U.S. Ser. Nos. 09/191,920;09/292,723; and 09/311,599 are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the process of microinjection of gene deliverymixture into a mammalian (mouse) testis. FIG. 1( a) shows a preferredsite of microinjection into a vas efferens; in mammals, the vasaefferentia connect to the lumen of all seminiferous tubules. FIG. 1( b)shows a vas efferens supported by a pipette tip, 1 mm diameter. FIG. 1(c) shows a mouse testis perfused with bromophenol blue after beinginjected in the vas efferens. FIG. 1( d) shows air bubbles in thetestis, confirming satisfactory delivery of viral particles.

FIG. 2 shows testicular cells transduced by a pseudotyped lentiviralvector expressing Green Fluorescent Protein (GFP) in Zeiss 410 confocalimages (wavelength 488 nm; 19 stacked images) of a cryosection of mousetestis. FIG. 2( a) shows a transduced Sertoli cell expressing GFP. FIG.2( b) shows transduced spermatogonia; GFP expression is visible in thecytoplasm surrounding large dark nuclei. FIG. 3 shows a DNA analysisfrom three consecutive litters of progeny from one male treated inaccordance with the in vivo method of incorporating exogenous geneticmaterial into the genome of a vertebrate. The top panel showsGFP-specific PCR amplification products separated on an agarose gel fromembryonic DNA of 22 individual progeny. In this run, there was anabsence of amplification from fetus No. 2, but other PCR assaysconfirmed the presence of the transgenic reporter gene. The bottom panelshows a Southern blot analysis of the same DNA. The Southern blot wasprobed with a radiolabed GFP DNA fragment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present method of incorporating exogenous genetic material into thegenome of a vertebrate relies on at least one of the followingstrategies. A first method, an in vivo method of incorporating exogenousgenetic material into the genome of a vertebrate, delivers apolynucleotide using known gene delivery systems to male germ cells insitu in the testis of the male vertebrate (e.g., in vivo transfection ortransduction), allows the genetically modified germ cells todifferentiate in their own milieu, and then selects for progeny animalsexhibiting the nucleic acid's integration into its germ cells(transgenic animals). The thus selected progeny can be mated, or theirsperm utilized for insemination or in vitro fertilization to producefurther generations of transgenic progeny.

Alternatively, the in vitro method of incorporating exogenous geneticmaterial into the genome of a vertebrate involves obtaining male germcells from the testis of a suitable donor or from the animal's owntestis, genetically modifying them in vitro, isolating or selectinggenetically modified germ cells, and then transferring them to the.testis under suitable conditions where they will spontaneouslyrepopulate it.

By either the in vivo or in vitro route, the inventive method issuitable for application to a variety of vertebrate animals, all ofwhich are capable of producing gametes, i.e. sperm or ova. Thus, inaccordance with the invention novel genetic modification(s) and/orcharacteristic(s) can be imparted to vertebrates, including mammals,such as humans, non-human primates, for example simians, marmosets,domestic agricultural animals such as ovines (e.g., sheep), bovines(e.g., cattle), porcines (e.g., pigs), equines (e.g., horses),particularly race horses, marine mammals, feral animals, rodents such asmice and rats, gerbils, hamsters, rabbits, and the like. Othervertebrate animals include fowl such as chickens, turkeys, ducks,ostriches, emus, geese, guinea fowl, doves, quail, rare and ornamentalbirds, and the like. Of particular interest are endangered species ofwild animal, such as rhinoceros, tigers, cheetahs, species of condor,and the like. “Gene delivery (or transfection) mixture”, in the contextof this patent, means selected genetic material together with anappropriate vector mixed, for example, with an effective amount of lipidtransfecting agent, for example, a cationic or polycationic lipid, suchas polybrene. (E.g., Clark et al., Polycations and cationic lipidsenhance adenovirus transduction and transgene expression in tumor cells,Cancer Gene Ther. 6(5):437-46 [1999]). The efficiency of adenoviral-,retroviral-, or lentiviral-mediated transduction is enhancedsignificantly by including polybrene during the infection. The amount ofeach component of the mixture is chosen so that the geneticmodification, e.g., by transfection or transduction, of a specificspecies of male germ cell is optimized. Such optimization requires nomore than routine experimentation. The ratio of DNA to lipid is broad,preferably about 1:1, although other proportions can also be utilizeddepending on the type of lipid transfecting agent used. (E.g., Baneijee,R. et al. [1999]; Jaaskelainen, I. et al., A lipid carrier with amembrane active component and a small complex size are required forefficient cellular delivery of anti-sense phosphorothioateoligonucleotides, Eur. J. Pharm. Sci. 10(3):187-193 [2000]; Sakurai, F.et al., Effect of DNA/liposome mixing ratio on the physicochemicalcharacteristics, cellular uptake and intracellular trafficking ofplasmid DNAlcationic liposome complexes and subsequent gene expression,J. Controlled Release 66(2-3):255-69 [2000]).

“Genetic material”, as used herein, means DNA sequences capable ofimparting novel genetic modification(s), or biologically functionalcharacteristic(s), to the recipient animal. The novel geneticmodification(s) or characteristic(s) can be encoded by one or more genesor gene segments defined by a polynucleotide, or can be caused byremoval or mutation of one or more genes, and can additionally containregulatory sequences, such as, but not limited to enhancers, promoters,or activator/suppressor binding sites. The transfected genetic materialis preferably functional, that is it expresses a desired trait by meansof a product or by suppressing the production of another. Examples ofother mechanisms by which a gene's function can be expressed are genomicimprinting, i.e. inactivation of one of a pair of genes (alleles) duringvery early embryonic development, or inactivation of genetic material bymutation or deletion of gene sequences, or by repression of a dominantnegative gene product, among others.

The desired product is any preselected product other than animmortalizing molecule, such as SV40 large T or polyoma virus large Tantigens. An immortalizing molecule can transform cells into“cancer-like” cells. “Immortalization” resulting from the expression ofan immortalizing molecule can cause a male germ cell to lose many of itsimportant germ cell characteristics, for instance the ability to undergomeiosis, which is crucial for the production of normally functioningmale gametes. (E.g., see, Wolkowicz, M. J., Coonrod, S. M., Reddi, P. P.Millan, J. L., Hofmann, M -C, Herr, J. C., Refinement of thedifferentiated phenotype of the spernatogenic cell line GC-2spd(ts),Biology of Reproduction 55:923-32 [1996]). Male germ cells geneticallymodified to express an immortalizing molecule are, therefore, not usefulfor the production of transgenic vertebrate progeny in accordance withthe present invention.

In addition, novel genetic modification(s) can be artificially inducedmutations or variations, or natural allelic mutations or variations of agene(s). Mutations or variations can be induced artificially by a numberof techniques, all of which are well known in the art, includingchemical treatment, gamma irradiation treatment, ultraviolet radiationtreatment, ultraviolet radiation, the use of specific chimeric DNA/RNAoligonucleotides (chimeraplasty), and the like. Chemicals useful for theinduction of mutations or variations include carcinogens such asethidium bromide and others known in the art.

DNA segments of specific sequences can also be constructed to therebyincorporate any desired mutation or variant or to disrupt a gene or toalter genomic DNA. Those skilled in the art will readily appreciate thatthe genetic material is inheritable and is, therefore, present in almostevery cell of future generations of the progeny, including the germcells. Among novel characteristics are the expression of a previouslyunexpressed trait, augmentation or reduction of an expressed trait, overexpression or under expression of a trait, ectopic expression, that isexpression of a trait in tissues where it normally would not beexpressed, or the attenuation or elimination of a previously expressedtrait. Other novel characteristics include the qualitative change of anexpressed trait, for example, to palliate or alleviate, or otherwiseprevent expression of an inheritable disorder with a multigenic basis.

“Transfecting agent”, as utilized herein, means a composition of matteradded to the genetic material for enhancing the uptake of exogenous DNAsegment(s) into a eukaryotic cell, preferably a mammalian cell, and morepreferably a mammalian germ cell. The enhancement is measured relativeto the uptake in the absence of the transfecting agent. Examples oftransfecting agents include adenovirus-transferrin-polylysine-DNAcomplexes. These complexes generally augment the uptake of DNA into thecell and reduce its breakdown during its passage through the cytoplasmto the nucleus of the cell. These complexes can be targeted to the malegerm cells using specific ligands which are recognized by receptors onthe cell surface of the germ cell, such as the c-kit ligand ormodifications thereof.

Other preferred transfecting agents include lipofectin, lipfectamine,DIMRIE C, Superfect, and Effectin (Qiagen), unifectin, maxifectin,DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP(1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB(NWN-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB(N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene, orpoly(ethylenimine) (PEI). (E.g., Banerjee, R. et al., Novel series ofnon-glycerol-based cationic transfection lipids for use in liposomalgene delivery, J. Med. Chem. 42(21):4292-99 [1999]; Godbey, W. T. etal., Improved packing of poly(ethylenimine/DNA complexes increasestransfection efficiency, Gene Ther. 6(8):1380-88 [1999]; Kichler, A etal., Influence of the DNA complexation medium on the transfectionefficiency of lipospermine/DNA particles, Gene Ther. 5(6):855-60 [1998];Birchaa, J. C. et al., Physico-chemical characterisation andtransfection efficiency of lipid-based gene delivery complexes, Int. J.Pharm. 183(2):195-207 [1999]). These non-viral agents have the advantagethat they facilitate stable integration of xenogeneic DNA sequences intothe vertebrate genome, without size restrictions commonly associatedwith virus-derived transfecting agents.

“Virus”, as used herein, means any virus, or transfecting fragmentthereof, which can facilitate the delivery of the genetic material intomale germ cells. Examples of viruses which are suitable for use hereinare adenoviruses, adeno-associated viruses, retroviruses such as humanimmune-deficiency virus, lentiviruses, mumps virus, and transfectingfragments of any of these viruses, and other viral DNA segments thatfacilitate the uptake of the desired DNA segment by, and release into,the cytoplasm of germ cells and mixtures thereof. A preferred viralvector is Moloney murine leukemia virus and the retrovirus vectorderived from Moloney virus calledvesicular-stomatitis-virus-glycoprotein (VSV-G)-Moloney murine leukemiavirus. A most preferred viral vector is a pseudotyped (VSV-G) lentiviralvector derived from the HIV virus (Naldini et al. [1996]). Also, themumps virus is particularly suited because of its affinity for immaturesperm cells including spermatogonia. All of the above viruses mayrequire modification to render them non-pathogenic or less antigenic.Other known vector systems, however, are also useful within the confinesof the invention.

In the in vivo method of incorporating exogenous genetic material intothe genome of a vertebrate, administering to a male vertebrate's testisa gene delivery mixture involves the in vivo introduction of the genedelivery mixture to the germ cells by direct delivery into at least oneof the animal's testes, where it is distributed to male germ cells atvarious stages of development. The in vivo method employs injection ofthe gene delivery mixture, preferably into the seminiferous tubules, orinto the rete testis, and most preferably into the vas efferens or vasaefferentia, using, for example, a micropipette. To ensure a steadyinfusion of the gene delivery mixture, under pressures which will notdamage the delicate tubule system in the testis, the injection is madethrough the micropipette with the aid of a picopump delivering a precisemeasured volume under controlled amounts of pressure. The micropipetteis made of a suitable material, such as, metal or glass, and is usuallymade from glass tubing which has been drawn to a fine bore at itsworking tip, e.g. using a pipette puller. The tip can be angulated in aconvenient manner to facilitate its entry into the testicular tubulesystem. Also, the micropipette can be provided with a beveled workingend to allow a better and less damaging penetration of the fine tubulesat the injection site. This bevel can be produced by means of aspecially manufactured grinding apparatus. The diameter of the tip ofthe pipette for the in vivo method of injection is typically about 15 to45 microns, although other sizes can be used as needed, depending on theanimal's size. The tip of the pipette can be introduced into the retetestis or the tubule system of the testicle, with the aid of a binocularmicroscope with coaxial illumination, with care taken not to damage thewall of the tubule opposite the injection point, and keeping trauma to aminimum. On average, a magnification of about 25× to 80× is suitable,and bench mounted micromanipulators are not severally required as theprocedure can be carried out by a skilled artisan without additionalaids. A small amount of a suitable, non-toxic dye, can be added to thegene delivery mixture (fluid) to confirm delivery and dissemination tothe seminiferous tubules of the testis. It can include a dilute solutionof a suitable, non-toxic dye, which can be visualized and tracked underthe microscope.

In this manner, the gene delivery mixture reaches and is brought intointimate contact with the male germ cells. Male germ cells includespermatozoa (i.e., male gametes) and developmental precursors thereof.In fetal development, primordial germ cells are thought to arise fromthe embryonic ectoderm, and are first seen in the epithelium of theendodermal yolk sac at the E8 stage. From there they migrate through thehindgut endoderm to the genital ridges. In the sexually mature malevertebrate animal, there are several types of cells that are precursorsof spermatozoa, and which can be genetically modified, including theprimitive spermatogonial stem cells, known as A0/As, which differentiateinto type B spermatogonia. The latter further differentiate to formprimary spermatocytes, and enter a prolonged meiotic prophase duringwhich homologous chromosomes pair and recombine. Useful precursor cellsat several morphological/developmental stages are also distinguishable:preleptotene spermatocytes, leptotene spermatocytes, zygotenespermatocytes, pachytene spermatocytes, secondary spermatocytes, and thehaploid spermatids. The latter undergo further morphological changesduring spermatogenesis, including the reshaping of their nucleus, theformation of acrosome, and assembly of the tail. The final changes inthe spermatozoan (i.e., male gamete) take place in the genital tract ofthe female, prior to fertilization. The polynucleotide contained in thegene delivery mixture administered in the in vivo method to the testiswill reach germ cells that are at any one of the above described stages,and will be taken up preferentially by those that are at a relativelymore receptive stage.

In the in vitro (ex vivo) method of incorporating exogenous geneticmaterial into the genome of a vertebrate, the male germ cells arepreferably, but not exclusively, diploid spermatogonia, which areexposed to or contacted with the gene delivery mixture.

Whether employed in the in vivo method or in vitro method, the genedelivery mixture, once in contact with the male germ cells, facilitatesthe uptake and transport of exogenous genetic material into theappropriate cell location for integration into the genome andexpression. A number of known gene delivery methods can be used for theuptake of nucleic acid sequences into the cell. In either the in vivo orvitro method, the gene delivery mixture typically comprises thepolynucleotide encoding the desired trait or product, together with asuitable promoter sequence, and optionally agents which increase theuptake of or comprise the polynucleotide sequence, such as liposomes,retroviral vectors, adenoviral vectors, adenovirus enhanced genedelivery systems, or combinations thereof. A reporter construct,including a genetic selection marker, such as the gene encoding forGreen Fluorescent Protein, can further be added to the gene deliverymixture. Targeting molecules, such as c-kit ligand, can be added to thegene delivery mixture to enhance the transfer of genetic material intothe male germ cell. An immunosuppressing agent, such as cyclosporin or acorticosteroid can also be added to the gene delivery mixture as knownin the art.

In the in vitro method of incorporating exogenous genetic material intothe genome of a vertebrate, the male germ cells are obtained orcollected from the donor male vertebrate, by means known in the art. Thethus obtained germ cells are then exposed to the gene delivery mixture,preferably within several hours, or cryopreserved for later use.

In one embodiment of the in vitro method, obtaining the male germ cellsfrom the donor vertebrate can be accomplished by transection of thetestes. Transection of the isolated testicular tissue can beaccomplished, for example, by isolation of the vertebrate's testes,decapsulation and teasing apart and mincing of the seminiferous tubules.The separated cells can then be incubated in an enzyme mixturecomprising enzymes known for gently breaking up the tissue matrix andreleasing undamaged cells such as, for example, pancreatic trypsin,collagenase type I, pancreatic DNAse type I, as well as bovine serumalbumin and a modified DMEM medium. The cells can be incubated in theenzyme mixture for a period of about 5 min to about 30 min, morepreferably about 15 to about 20 min, at a temperature of about 33° C. toabout 37° C., more preferably about 36 to 37° C. After washing the cellsfree of the enzyme mixture, they can be placed in an incubation mediumsuch as DMEM, and the like, and plated on a culture dish for geneticmodification by exposure to the gene delivery mixture. This transectionmethod is not suitable when the donor and recipient male vertebrates areintended to be the same animal, in which case induced a less destructivebiopsy method or induced ejaculation by means known in the art ispreferred.

Any of a number of commercially available gene delivery mixtures can beused, to which the polynucleotide encoding a desire trait or product isfurther admixed. The final gene delivery mixture comprising thepolynucleotide can then be admixed with the cells and allowed tointeract for a period of about 2 hrs to about 16 hrs, preferably about 3to 4 hrs, at a temperature of about 33° C. to about 37° C., preferablyabout 36° C. to 37° C., and more preferably in a constant and/orcontrolled atmosphere. After this period, the cells are preferablyplaced at a lower temperature of about 33° C. to about 34° C.,preferably about 30-35° C. for a period of about 4 hrs to about 20 hrs,preferably about 16 to 18 hrs. Other conditions which do not deviateradically from the ones described can a also be utilized as an artisanwould know.

With respect to either the in vivo or in vitro methods, a most preferredembodiment employs a retroviral vector system, which was developed forgene therapy (Naldini, L., et al., In vivo gene delivery and stabletransduction of nondividing cells by a lentiviral vector, Science 272:263-267 [1996]), which is used to transduce male germ cells in vivo orin vitro. This gene delivery system employs retroviral particlesgenerated by a three-plasmid expression system. In this system apackaging construct contains the human cytomegalovirus (hCMV) immediateearly promoter, driving the expression of all viral proteins. Theconstruct's design eliminates the cis-acting sequences crucial for viralpackaging, reverse transcription and integration of these transcripts.The second plasmid encodes a heterologous envelope protein (env), namelythe G glycoprotein of the vesicular stomatitis virus (VSV-G). The thirdplasmid, the transducing vector (pHR′), contains cis-acting sequences ofhuman immunodeficiency virus (HIV) required for packaging, reversetranscription and integration, as well as unique restriction sites forcloning heterologous complementary DNAs (cDNAs). For example, a geneticselection marker, such as the enhanced green fluorescent protein (EGFP),and/or a gene encoding another preselected or desired trait or productis cloned downstream of the hCMV promoter in the HR'vector, and isoperatively linked so as to form a transcriptional unit. A VSV-Gpseudotyped retroviral vector system is capable of infecting a widevariety of cells including cells from different species and ofintegrating into the genome. Some retroviruses, i.e., lentiviruses, suchas HIV, have the ability to infect non-dividing cells. They have alimited capacity for heterologous DNA sequences, the size limit for thisvector being 7-7.5 kilobases (Verma, I. M. and Somia, N., GeneTherapy—promises, problems and prospects, Nature 389:239-242 [1997]). Invivo experiments with lentiviruses show that expression does not shutoff like other retroviral vectors and that in vivo expression in brain,muscle, liver or pancreatic-islet cells, is sustained at least for oversix months—the longest time tested so far (Verma and Somia [1997];Anderson, W F., Human Gene Therapy, Nature (Suppl). 392:25-30 [1998]).

For the expression of delivered genetic material by transfection,transduction, or other means to obtain expression of a desired trait orproduct, a promoter sequence is operatively linked to apolynucleotidesequence encoding the desired trait orproduct. For purposes of thepresent invention, “operatively linked” means that, within atranscriptional unit, the promoter sequence, is located upstream (i.e.,5′ in relation thereto) from the coding sequence and the codingsequence, is 3′ to the promoter, or alternatively is in a sequence ofgenes 3′ to the promoter and expression is coordinately regulatedthereby. Both the promoter and coding sequences are oriented in a 5′ to3′ manner, such that transcription can take place in vitro in thepresence of all essential enzymes, transcription factors, co-factors,activators, and reactants, under favorable physical conditions, e.g.,suitable pH and temperature. This does not mean that, in any particularcell, conditions will favor transcription. For example, transcriptionfrom a tissue-specific promoter is generally not favored in heterologouscell types from different tissues.

A promoter sequence is chosen that operates in the cell type of interestand/or under the physiologic or developmental conditions of interest.Useful promoter sequences include constitutive promoters, such as, butnot limited to, cytomegalovirus (CMV) promoter, or inducible promoters,such as, but not limited to, the human C-reactive protein (CRP) promoter(e.g., Kanzler, S., et al., TGF-betal in liver fibrosis: an inducibletransgenic mouse model to study liver fibrogenesis, Am. J. Physiol.276(4Pt 1):G1059-68 [1999]), or the insulin-like growth factor (IGF-I)promoter (e.g., Meton I., et al., Growth hormone induces insulin-likegrowth factor-I gene transcription by synergistic action of STAT5 andHNF-1alpha, FEBS Lett. 444(2-3):155-59 [1999]). Useful promoters includethose that promote transcription in cells of diverse tissues, such as,but not limited to, an insulin receptor (IR) gene promoter (e.g.,Tewari, D. S., et al., Characterization of the promoter region and 3′endof the human insulin receptor gene, J. Biol. Chem. 264(27):16238-45[1989]); growth hormone receptor (GHR) P2 or P3 promoters (e.g., Jiang,H., et al., Isolation and characterization of a novel promoterfor thebovine growth hormone receptor gene, J. Biol. Chem. 274(12):7893-900[1999]); or a leptin promoter (e.g., Chen, X. L., et al., Analysis of a762-bp proximal leptin promoter to drive and control regulation oftransgene expression of growth hormone receptor in mice, Biochem.Biophys. Res. Commun. 262(1):187-92 [1999]).

Also useful for various applications are tissue-selective (i.e.,tissue-specific) promoters, i.e., promoters from which expression occurspreferentially in cells of a particular kind of tissue, compared to oneor more other types of tissue. Tissue-specific promoters areparticularly useful in applications directed to gene therapy or to thegenetic enhancement of non-human vertebrates.

For example, a promoter sequence, which is only active in cyclingspermatogonial stem cell populations can be used for differentialexpression in male germ cells, for example, B-Myb or a male germcell-specific promoter, such as the c-kit promoter region, c-raf-1promoter, ATM (ataxia-telangiectasia) promoter (also active incerebellar cells and thymocytes), vasa promoter, cyclin Alpromoter, RBM(ribosome binding motif) promoter, DAZ (deleted in azoospermia)promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, or FRMI(from fragile X site) promoter.

For hematopoietic tissue-selective expression in hematopoietic precursorcells, useful promoters include cyclin Al promoters (e.g., Müller, C.,et al., Cloning of the cyclin A1 genomic structure and characterizationof the promoter region, J. Biol. Chem. 276(16):11220-28 [1999]); CD34promoters (e.g., Burn, T. C., et al., Hematopoietic stem cell specificgene expression, U.S. Pat. No. 5,556,954); a c-kit promoter, or anintegrin alphaIIb promoter (e.g., Wilcox, D. A., et al., IntegrinalphaIIb promoter-targeted expression of gene products in megakaryocytesderived from retrovirus-transduced human hematopoietic cells, Proc.Natl. Acad. Sci. USA 96(17):9654-59 [1999]).

Cartilage-selective promoters for expression in chondrocytes, forexample, an osteocalcin (OC) promoter (e.g., Newberry, E. P., et al.,The RRM domain of MINT, a novel Msx2 binding protein, recognizes andregulates the rat osteocalcin promoter, Biochemistry 38(33):10678-90[1999]); a SOX9 promoter, aggrecan gene promoter (AGC1), or collagenoligomeric matrix protein (COMP) gene promoter (e.g., Kanai, Y. &Koopman, P., Structural and functional characterization of the mouseSox9 promoter: implications for campomelic dysplasia, Hum. Mol. Genet.8(4):691-96 [1999]; Newton et al., Characterization of human and mousecartilage oligomeric matrix protein, Genomics 24:435-39 [1994]; or apromoter from a collagen gene, such as, but not limited to promoters forCOL2A1, COL9A1, or COL10A1. (e.g., Ganguly, A., et al., Targetedinsertions of two exogenous collagen genes into both alleles of theirendogenous loci in cultured human cells: the insertions are directed byrelatively short fragments containing the promoters and the 5′ ends ofthe genes, Proc Natl Acad Sci USA 91(15):7365-9 [1994]; Dharmavaram, R.M., et al., Detection and characterization of Sp1 binding activity inhuman chondrocytes and its alterations during chondrocytededifferentiation, J. Biol Chem 272(43):26918-25 [1997]; Zhou, G., etal., Three high mobility group-like sequences within a 48-base pairenhancer of the Col2a1 gene are required for cartilage-specificexpression in vivo, J. Biol Chem 273(24):14989-97 [1998];Seghatoleslami, M. R., et al., Differential regulation of COL2A1expression in developing and mature chondrocytes, Matrix Biol14(9):753-64 [1995]; Lefebvre, V., et al., An 18-base-pair sequence inthe mouse proalphal(II) collagen gene is sufficient for expression incartilage and binds nuclear proteins that are selectively expressed inchondrocytes, Mol Cell Biol 16(8):4512-23 [1996]; Zhou, G., et al., A182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficientto direct chondrocyte expression in transgenic mice, J. Cell Sci, 108(Pt12):3677-84 [3677-84]; Mukhopadhyay, K., et al., Use of a new ratchondrosarcoma cell line to delineate a 119-base pairchondrocyte-specific enhancer element and to define active promotersegments in the mouse pro-alpha 1(II) collagen gene, J. Biol Chem270(46):27711-9 [1995]; Vikkula, M., et al., Structural analysis of theregulatory elements of the tyupe-II procollagen gene. Conservation ofpromoter and first intron sequences between human and mouse, Biochem J285(Pt 1):287-94 [1992]; Beier, F., et al., Localization of silencer andenhancer elements in the human type X collagen gene, J Cell Biochem662(2):210-8 [1997]; Thomas, J. T., Sequence comparison of threemammalian type-X collagen promoters and preliminary functional analysisof the human promoter, Gene 160(2):291-6 [1995]; Apte, S. S.,Characterization of the mouse type X collagen gene, Matrix 13(2):165-79[1993]). A cartilage-derived retinoic acid-sensitive protein (CD-RAP)gene promoter is also useful for cartilage-selective expression bychondrocytes. (e.g., Xie, W. F., et al., Transactivation of the mousecartilage derived retinoic acid-sensitive protein gene by Sox9, J. BoneMiner. Res. 14(5):757-63 [1999]).

For liver-selective expression in hepatocytes, useful promoter sequencesinclude, an albumin gene promoter (e.g., Pastore, L., et al., Use of aliver-specific promoter reduces immune response to the transgene inadenoviral vectors, Hum. Gen. Ther. 10(11):1773-81 [1999]); a CYP7A orCYP7A1 promoter (e.g., Nitta, M., et al., CPF: an orphan nuclearreceptor that regulates liver-specific expression of the humancholesterol 7alpha-hydroxylase gene, Proc. Natl. Acad. Sci. USA96(12):6660-65 [1999]; Chen, J., et al., Hepatocyte nuclear factor 1binds to and transactivates the human but not the rat CYP7A1 promoter,Biochem. Biophys. Res. Commun. 260(3):829-34 [1999] ); a GHR P1 promoter(e.g., Zou, L., et al., Isolation of a liver-specific promoter for humangrowth hormone receptor gene, Endocrinology 138(4):1771-74 [1997];Jiang, H., et al. [1999]; Adams, T. E., Differential expression ofgrowth hormone receptor messenger RNA from a second promoter, Mol. CellEndocrinol. 108(1-2):23-33 [1995]); or a thrombin-activatablefibrinolysis inhibitor (TAFI) promoter (e.g., Boffa, M. B., et al.,Characterization of the gene encoding human TAFI [thrombin-activatablefibrinolysis inhibitor; plasma procarboxypeptidase B], Biochemistry38(20):6547-58 [1999]).

Neuronal specific promoters are also useful, for example, aneurofilament promoter or a neural-specific enolase promoter.

Many other tissue specific promoters are useful for tissue specificexpression of a preselected gene for phenotypic expression of a desiredtrait or product in the various tissues or organs of the vertebratebody.

Other useful promoters are related to the expression ofcytokine-inducible proteins, including promoters that regulate theexpression of products and modulators of the Jak-STAT signaling cascade,for example, a SOCS-3 promoter (C. J. Auernhammer et al., Autoregulationof pituitary corticotroph SOCS-3 expression: characterization of themurine SOCS-3 promoter, Proc. Natl. Acad Sci. USA 96:6964-69 [1999]), aSTAT-3 promoter (C. Bousquet & S. Melmed, J. Biol. Chem. 274:10723-30[1999]), a POMC promoter (C. J. Auernhammer et al. [1998b]), or Spi 2.1promoter (T. E. Adams et al. [1995]).

Useful promoters also include exogenously inducible promoters. These arepromoters that can be “turned on” in response to an exogenously suppliedagent or stimulus, which is generally not an endogenous metabolite orcytokine. Examples include an antibiotic-inducible promoter, such as atetracycline-inducible promoter; a heat-inducible promoter; alight-inducible promoter; or a laser-inducible promoter. (E.g.,Halloran, M. C. et al., Laser-induced gene expression in specific cellsof transgenic zebrafish, Development. 127(9):1953-1960 [2000]; Gemer, E.W. et al., Heat-inducible vectorsfor use in gene therapy, Int. J.Hyperthermia 16(2):171-81 [2000]; Rang, A., and Will, H., Thetetracycline-responsive promoter contains functionalinterferon-inducible response elements, Nucleic Acids Res. 28(5): 1120-5[2000]; Hagihara Y. et al., Long-term functional assessment ofencapsulated cells transfected with Tet-On system, Cell Transplant.8(4):431-4 [1999]; Huang, C. J. et al., Expression of green fluorescentprotein in oligodendrocytes in a time-and level-controllable fashionwith a tetracycline-regulated system, Mol. Med. 5(2):129-37 [1999];Forster, K. et al., Tetracycline-inducible expression systems withreduced basal activity in mammalian cells, Nucleic Acids Res.27(2):708-10 [1999]; Liu, H. S. et al., Lac/Tet dual-inducible systemfunctions in mammalian cell lines, Biotechniques 24(4):624-8, 630-2[1998]).

Other useful promoters include developmentally or temporally regulatedpromoters. Examples include the myelin P0 promoter (P. Thatikunta etal., Reciprocal Id expression and myelin gene regulation in Schwanncells, Mol. Cell Neurosci. 14(6):519-28 [1999]), Gabra3 or GABRA3promoters (W. Mu and D. R. Burt, the mouse GABA(A) receptor alpha3subunit gene and promoter, Brain Res. Mol. Brain Res. 73(1-2):172-80[1999]), tyrosine hydroxylase promoter (J. J. Schimmel et al., 4.5 kb ofthe rat tyrosine hydroxylase 5′ flanking sequence directs tissuespecific expression during development and contains consensus sites formultiple transcription factors, Brain Res. Mol. Brain Res. 74(1-2): 1-14[1999]), vimentin promoters (A. Benazzouz and P. Duprey, The vimentinpromoter as a tool to analyze the early events of retinoic acid-induceddifferentiation of cultured embryonal carcinoma cells, Differentiation(65(3):171-80 [1999]), GATA-6 promoters (A. Brewer et al., The human andmouse GATA-6 genes utilize two promoters and two initiation codons, J.Biol. Chem. 274(53):38004-16 [2000]), SHIP1 or SHIP2 promoters (S.Schurmans et al., The mouse SHIP2 (Inppll)gene: complementary DNA,genomic structure, promoter analysis, and gene expression in the embryoand adult mouse, Genomics 62(2):260-71 [1999]), or hGH-N promoters (B.M. Shewchuk et al., Pit-I binding sites at the somatotrope-specificDNase I hypersensitive sites I, II, of the human growth hormone locuscontrol region are essential for in vivo hGH-N gene activation, J. Biol.Chem. 274(50):35725-33 [1999]).

The foregoing examples of useful promoter sequences are by no means anexhaustive list, but are merely illustrative of the promoters availableto the skilled artisan in practicing the present invention.

The in vivo and in vitro methods of incorporating exogenous geneticmaterial into the genome of a vertebrate involve incorporating thepolynucleotide encoding a desired trait or product into the genome of atleast one spermatozoan or precursor thereof, so that a geneticallymodified male gamete is produced by the male vertebrate. Thus, thegenetically modified germ cells of the vertebrate animal, nowtransgenic, have the non-endogenous (exogenous) genetic materialintegrated into their chromosomes. This is what is often referred to asa “stable transfection” or “stable integration”. This is applicable toall vertebrate animals, including humans. Animals that are shown tocarry suitably modified sperm cells then can be either allowed to matenaturally, or alternatively their spermatozoa are used for inseminationor in vitro fertilization.

Isolating and/or selecting of genetically modified cells, includingtransgenic germ cells and transgenic somatic cells, and of transgenicvertebrates, is by any suitable means, such as, but not limited to,physiological and/or morphological phenotypes of interest using anysuitable means, such as biochemical, enzymatic, immunochemical,histologic, electrophysiologic, biometric or like methods; and analysisof cellular nucleic acids, for example the presence or absence ofspecific DNAs or RNAs of interest using conventional molecularbiological techniques, including hybridization analysis, nucleic acidamplification (such as but not limited to, polymerase chain reaction[PCR], reverse transcriptase-mediated polymerase chain reaction[RT-PCR], transcription-mediated amplification [TMA], reversetranscriptase-mediated ligase chain reaction [RT-LCR]), and/orelectrophoretic technologies.

In a preferred embodiment, the gene delivery mixture includes at leastone polynucleotide comprising a gene encoding a genetic selection markerthat is operatively linked to a promoter sequence such that atranscriptional unit is formed. The promoter sequence can be the same ordifferent from the promoter regulating the expression from the geneencoding the desired trait or product. The genetic selection marker,also known as a reporter gene, is, for example, a gene encoding anenzyme, such as P-galactosidase, or encoding a fluorescent protein, suchas Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein(EGFP), Yellow Fluorescent Protein, Blue Fluorescent Protein, aphycobiliprotein, such as phycoerythrin or phycocyanin, or any otherprotein which fluoresces under a suitable wave-length.

Another preferred genetic selection marker or reporter gene, suitablefor some applications is a gene encoding a protein that canenzymatically lead to the emission of light from a substrate(s); forpurposes of the present invention, such a protein is a “light-emitting”or luminescent protein. For example, a light-emitting protein includesproteins such as luciferase or apoaequorin. Transgenic cells expressinga fluorescent or luminescent protein encoded by the reporter constructcan be sorted with the aid of, for example, a flow activated cell sorter(FACS) set at the appropriate wavelength(s), or they can be selected bychemical methods.

A preferred method of isolating or selecting male germ cell populations,comprises obtaining specific male germ cell populations, such asspermatogonia, from a mixed population of testicular cells by extrudingthe cells from the seminiferous tubules and gentle enzymaticdisaggregation. The spermatogonia or other male germ cell populations,which are to be genetically modified, can be isolated from a mixed cellpopulation by a method including the utilization of a promoter sequence,which is specifically or selectively active in cycling male germ linestem cell populations, for example, B-Myb or a specific promoter, suchas the c-kit promoter region, c-raf-1 promoter, ATM(ataxia-telangiectasia) promoter, vasa promoter, RBM (ribosome bindingmotif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter,HSP 90 (heat shock gene) promoter, cyclin A1 promoter, or FRMI (fromfragile X site) promoter, linked to a reporter construct, for example, aconstruct comprising a gene encoding Green Fluorescent Protein (orEGFP), Yellow Fluorescent Protein, Blue Fluorescent Protein, aphycobiliprotein, such as phycoerythrin or phycocyanin, or any otherprotein which fluoresces under suitable wave-lengths of light, orencoding a light-emitting protein, such as luciferase or apoaequorin.These unique promoter sequences drive the expression of the reporterconstruct only during specific stages of male germ cell development, asis known to those skilled in the art. (E.g., Müller, C., et al., Cloningof the cyclin A1 genomic structure and characterization of the promoterregion, J. Biol. Chem. 276(16):11220-28 [1999]; Schrans-Stassen, B., H.et al., Differential expression of c-kit in mouse undifferentiated anddifferentiating type A spermnatogonia, Endocrinology 140:5894-5900[1999]). Populations of male germ cells at specific developmentalstages, thus, are the only cells in the mixed population which willexpress the reporter construct(s) and they, thus, can be isolated onthis basis. In the case of a fluorescent reporter construct, the cellscan be sorted with the aid of, for example, a FACS set at theappropriate wavelength(s), or they can be selected by chemical methods.

Further with respect to the in vitro method of incorporating exogenousgenetic material into the genome of a vertebrate, in which male germcells are obtained from a donor animal and genetically modified in vitroto impart a gene encoding a desired trait or product, male germ cellswhich exhibit any evidence that the DNA has been modified in the desiredmanner are isolated or selected, and transferred to the testis of asuitable recipient animal. After transfer, further selection can beattempted after biopsy of one or both of the recipient male's testes, orafter examination of the animal's ejaculate amplified by the polymerasechain reaction to confirm whether the desired nucleic acid sequence wasactually incorporated. As described above, the initial gene delivery canhave included a reporter gene, such as a gene encoding the GreenFluorescent Protein, enhanced Green Fluorescent Protein (EGFP), YellowFluorescent Protein, Blue Fluorescent Protein, a phycobiliprotein, suchas phycoerythrin or phycocyanin, or any other protein which fluorescesunder light of suitable wave-lengths, or encoding a light-emittingprotein.

In the in vitro method of incorporating exogenous genetic material intothe genome of a vertebrate, the genetically modified germ cells, thusisolated or selected, are preferably transferred to a testis of arecipient male vertebrate, which can be, but need not be, the same donoranimal. Before transferring the genetically modified male germ cells toone or more of the testes of the recipient male vertebrate, the testesof the recipient animal are preferably depopulated of native germ cells.

Substantial depopulation of the endogenous male germ cells facilitatesthe colonization of the recipient testis by the genetically modifiedgerm cells from the donor animal. The depopulation can be done by anysuitable means, including by gamma irradiation, by chemical treatment,by means of infectious agents such as viruses, or by autoimmunedepletion or by combinations thereof.

Whichever means of depopulating the testis of endogenous male germ cellsis used, the basic rigid architecture of the gonad should not bedestroyed, nor badly damaged. If there is disruption of the fine systemof tubule formation, it may be impossible for the exogenousspermatogonia to repopulate the testis. Disruption of tubules would alsopresumably lead to impaired transport of testicular sperm and result ininfertility. Any controlled testicular injury of this kind should alsobe limited so that the Sertoli cells are not irreversibly damaged, asthey are needed to provide a base for development of the germ cellsduring maturation. Moreover they may play a role in preventing the hostimmune defense system from destroying grafted foreign spermatogonia.

But vertebrate testes are most preferably depopulated by a combinedtreatment of the vertebrate with an alkylating agent and gammairradiation in accordance with the inventive method of substantiallydepopulating a vertebrate testes. The method involves a treatment with acytotoxic alkylating agent, such as, but not limited to, busulfan(1,4-butanediol dimethanesulphonate; Myleran, Glaxo Wellcome),chlorambucil, cyclophosphamide, melphalan, or ethyl ethanesulfonic acid,combined with gamma irradiation, to be administered in either sequence.The combination of a dose of an alkylating agent and a dose of gammaradiation yields unexpectedly superior results in depopulating thetestes of germ cells, compared to either treatment alone. The dose ofthe alkylating agent and the dose of gamma radiation are in an amountsufficient to substantially depopulate the testis.

The preferred dose of alkylating agent is about 4 to 10 milligrams perkilogram of body weight, and about 6 to 8 milligrams per kilogram ofbody weight is most preferred. The alkylating agent can be administeredby any pharmaceutically acceptable delivery system, including but notlimited to, intraperitoneal, intravenous, or intramuscular injection,intravenous drip, implant, transdermal or transmucosal delivery systems.

A recovery period between the administration of alkylating agent andirradiation is not essential, and the two treatments are most preferablydone within zero to 24 hours of each other. Preferably, the time betweenthe two treatments should not exceed 2 weeks, because this yields lessthan optimal results for purposes of transferring genetically modifiedor heterologous male germ cells to recipient testes.

The recipient vertebrate is gamma irradiated with a dose of about 200 to800 Rads, most preferably about 350 to 450 Rads, directed locally to thetestis to be depopulated. Less than 200 Rad yields little effect;greater than 800 Rad commonly produces symptoms of radiation sickness,particularly in the gastrointestinal tract. Within 3 days to 2 monthsafter treatment to depopulate the recipient testis(es) in accordancewith the present method, male germ cells can be transferred thereto asdescribed herein. Prior to three days, traces of cytotoxic alkylatingagent or endogenous apoptotic signal molecules may remain in therecipient testis to harm the male germ cells transferred thereto. Aftertwo months, the endogenous population of male germ cells will typicallybegin to restablish itself, yielding less than optimal results whentransfected, genetically altered, or heterologous male germ cells aretransferred to a recipient testes for breeding purposes.

Thus in the in vitro (ex vivo) method, preferably within three days totwo months after the final treatment to depopulate the testis(es) of therecipient male vertebrate, the gene delivery mixture is administered tothe male germ cells of the donor vertebrate, in vitro, in sufficientamount and under effective conditions such that one or more of them isgenetically modified. Genetically modified male germ cells from thedonor male vertebrate can then be transferred to the testis(es) of therecipient male such that they lodge in a seminiferous tubule of thetestis, where they then mature into genetically modified gametes.

Transferring the isolated or selected genetically modified germ cellsinto the recipient testis can be accomplished by direct injection usinga suitable micropipette. Support cells, such as Leydig or Sertoli cellsthat provide hormonal stimulus to spermatogonial differentiation, can betransferred to a recipient testis along with the modified germ cells.These transferred support cells can be unmodified, or, alternatively,can themselves have been genetically modified, together with—orseparately from the germ cells. These transferred support cells can beautologous or heterologous to either the donor or recipient testis. Apreferred concentration of cells in the transfer fluid can easily beestablished by simple experimentation, but will likely be within therange of about 1×10⁵−10×10⁵ cells per 10 μl of fluid. This micropipettecan be introduced into the vasa efferentia, the rete testis or theseminiferous tubules, optionally with the aid of a picopump to controlpressure and/or volume, or this delivery can be done manually. Themicropipette employed is in most respects similar to that used for thein vivo injection, except that its tip diameter generally will be about45 to about 70 microns. The microsurgical method of introduction issimilar in all respects to that used for the in vivo method describedabove. A suitable dyestuff or bubbles (less than 1 mm in diameter) canalso be incorporated into the carrier fluid for easy identification ofsatisfactory delivery of the transfected germ cells to at least oneseminiferous tubule of the testis (FIG. 1).

With respect to both the in vivo and in vitro methods of incorporatingexogenous genetic material into the genome of a vertebrate involves,breeding the male vertebrate with a female vertebrate of its speciesmeans causing the union of male and female gametes so that fertilizationoccurs and a transgenic zygote is formed; a transgenic progeny oroffspring is thereafter produced during gestation of the developingfetus. A union of male and female gametes is brought about by naturalmating, i.e., copulation by the male and female vertebrates of the samespecies, or by in vitro or in vivo artificial means. If artificial meansare chosen, then incorporating into the genome a genetic selectionmarker that is expressed in male germ cells is particularly useful.Preferably expression of the genetic selection marker is regulated froma constitutive or male germ-cell specific promoter, operatively linkedto the gene encoding the genetic selection marker.

Artificial means include, but are not limited to, artificialinsemination, in vitro fertilization (IVF) and/or other artificialreproductive technologies, such as intracytoplasmic sperm injection(ICSI), subzonal insemination (SUZI), or partial zona dissection (PZD).However, others, such as cloning and embryo transfer, cloning and embryosplitting, and the like, can also be employed.

The thus obtained transgenic vertebrate progeny can, in turn, also bebred, whether by natural mating, artificial insemination, or by in vitrofertilization (IVF) and/or other artificial reproductive technologies,such as intracytoplasmic sperm injection (ICSI), subzonal insemination(SUZI), or partial zona dissection (PZD), to obtain further generationsof transgenic progeny. Those skilled in the art will readily appreciatethat any desired traits generated as aresult of changes to the geneticmaterial of any transgenic animal produced by the inventive method areinheritable. Although the genetic material was originally insertedsolely into the germ cells of a parent animal, it will ultimately bepresent in the germ cells of future progeny and subsequent generationsthereof. In addition, the genetic material is also present in cells ofthe progeny other than germ cells, i.e., somatic cells.

Broadly speaking, a “transgenic” vertebrate is one that has had foreignor exogenous DNA permanently introduced into its cells. The exogenousgenes which have been introduced into the animal's cells are called“transgenes” and are xenogeneic and/or allogeneic transgenic geneticmaterial, including biologically functional genetic material. Thepresent invention is applicable to the production of transgenic animalscontaining xenogeneic, i.e., exogenous DNA from a different species,either in its native, undisturbed form, or in artificially mutated form.In other embodiments, the genetic material is “allogeneic” geneticmaterial, exogenous transgenic material obtained from a differentstrain, race, breed, or individual of the same species, for example,from an animal having a “normal” form of a gene, or a desirable allele,variant, or mutation thereof. Also the gene can be a hybrid constructconsisting of promoter DNA sequences and DNA coding sequencesoperatively linked together. These sequences can be obtained fromdifferent species or DNA sequences from the same species that are notnormally juxtaposed. The DNA construct can also contain DNA sequencesfrom prokaryotic organisms, such as bacteria, or from viruses.

The transfected germ cells of the transgenic vertebrate animalpreferably have the non-endogenous (exogenous) genetic materialintegrated into their chromosomes. Those skilled in the art will readilyappreciate that any desired traits generated as a result of changes tothe genetic material of any transgenic vertebrate produced by thisinvention are heritable. Although the genetic material was originallyinserted solely into the germ cells of a parent animal, it willultimately be present in the germ cells of direct progeny and subsequentgenerations thereof. The genetic material is also present in thedifferentiated cells, i.e. somatic cells, of the progeny.

Included in the invention is a non-human transgenic male vertebrateproduced by the in vivo or in vitro method of incorporating exogenousgenetic material into the genome of a vertebrate. Produced in accordancewith the in vivo method, the transgenic. vertebrate is the recipient ofthe gene delivery mixture. Alternatively, the non-human transgenic malevertebrate is the recipient of the genetically modified male germ cellthat was transferred to its testis, in accordance with the in vitromethod. The transgenic male vertebrate can be bred with a female of itsspecies, because it comprises a native male germ cell carrying in itsgenome a polynucleotide of exogenous origin defining a gene encoding adesired trait or product. But somatic cells in tissues outside thetestis of the transgenic vertebrate lack the polynucleotide. Preferably,but not necessarily, the transgenic male vertebrate will continue toproduce genetically modified gametes for an indefinite period. However,in some embodiments the transgenic state is temporary, lasting for atleast several weeks or months, after which non-modified gametes areagain exclusively or predominantly produced by the animal.

Also included in the invention is a non-human transgenic vertebrateproduced in accordance with the in vivo or in vitro method ofincorporating exogenous genetic material into the genome of avertebrate, wherein the non-human vertebrate is the direct or indirectprogeny of the transgenic male vertebrate described above. Thus, thistransgenic progeny is the immediate offspring, male or female, of thetransgenic male vertebrate or is an offspring thereof separated by oneor more generations. The transgenic vertebrate includes one or morecells carrying in their genome a polynucleotide of exogenous origin thatencodes a desired trait or product.

Also, the invention includes a transgenic cell derived from thetransgenic vertebrate progeny. The cell is a germ cell, such as aspermatozoan or ovum, a precursor germ cell of either of these, or asomatic cell.

Male germ cells are obtained from a male animal's semen, or spermatozoa,spermatogonia, or immature spermatocytes are separated from wholebiopsies of testicular tissue containing the male germ cells.Alternatively, male germ line stem cells can be isolated from embryonictissue. Female germ cells are obtained by known means, includinghormonally induced “ripening” and harvesting from the oviducts oraspiration by way of the cervix or by way of a laparoscopic incision.

Somatic cells include stem cells. A stem cell is an undifferentiatedmother cell Ithat is self-renewable over the life of the organism and ismultipotent, i.e., capable of generating various committed progenitorcells that can develop into fully mature differentiated cell lines.(E.g., T. Zigovaand P. R. Sanberg, The rising star of neural stem cellresearch, Nature Biotechnol. 16(11):1007-08 [1998]). All vertebratetissues arise from stem cells, including hematopoietic stem cells, fromwhich various types of blood cells derive; ectodermal stem cells; neuralstem cells, for example, neural progenitors from which brain and nervetissues derive. Somatic cells also include progenitor cells orterminally differentiated cells of any kind associated with any tissueor organ of the vertebrate body.

Somatic cells are obtained by known sampling or biopsy means from anybodily tissue, organ, or fluid, including but not limited to, blood,heart, kidney, ureter, bladder, urethra, brain, thyroid, parotid gland,pancreas, hypothalamus, pituitary gland, submaxillary gland, sublingualgland, lymph node, bone, bone marrow, cartilage, lung, mediastinum,breast, uterus, ovary, testis, prostate, cervix uteri, endometrium,liver, spleen, adrenal, esophagus, stomach, intestine, hair root,muscle, nerve, urine, amniotic fluid, chorionic villus, skin, vascularor oral epithelium, or spinal fluid. The inventive transgenic cells canbe cultured or stored by well known means.

The invention also relates to vertebrate semen containing a plurality ofthe inventive transgenic male germ cell. The inventive vertebrate semenis useful for breeding or other suitable purposes. The semen is obtainedfrom ejaculate produced by the inventive transgenic male vertebrateorits transgenic male progeny (either immediate progeny or progenyseparated by one or more generations), and methods of inducingejaculation by a male vertebrate and capturing the semen are well known.The semen can be processed, e.g., by washing, and/or stored by meanssuch as are known in the art. For example, storage conditions includethe use of cryopreservation using programmed freezing methods and/or theuse of cryoprotectants, for example, dimethyl sulfoxide (DMSO),glycerol, trehalose, or propanediol-sucrose, and the use of storage insubstances such as liquid nitrogen.

Such storage techniques are particularly beneficial to young adulthumans or children, undergoing oncological treatments for such diseasessuch as leukemia or Hodgkin's lymphoma. These treatments frequentlyirreversibly damage the testicle and, thus, render it unable torecommence spermatogenesis after therapy by, for example, irradiation orchemotherapy. In species other than humans, the present techniques arevaluable for transport of gametes as frozen germ cells. Such transportwill facilitate the establishment of various valued livestock or fowllines, at a remote distance from the donor animal. This approach is alsoapplicable to the preservation of endangered species across the globe.

Thus, the invention is also includes a method of producing a non-humantransgenic vertebrate animal line, the individuals of which comprisenative germ cells carrying in their genome at least one xenogeneicpolynucleotide. The transgenic vertebrates bred with other transgenic ornon-transgenic animals of the same species will produce some transgenicprogeny, including fertile individuals. The method involves breeding ofthe fertile transgenic progeny with a member of the opposite sex of thesame species as described above; and selecting its progeny for thepresence of the polynucleotide. The inventive method of producing anon-human transgenic vertebrate animal line is simple and efficient, andis more easily accomplished in large mammals than in mice because of thelarger size of the testicular ducts. Far fewer animals are needed toproduce transgenic progeny by genetic modification of male germ cells,which can be produced continually from repeated mating withoutinterruption by pregnancy or parturition. It requires no expensiveequipment, nor the training necessary for microinjection.

The inventive technology is applicable to the field of gene therapy,since it permits the introduction of genetic material encoding andregulating specific genetic traits. Thus, in the human, for example, bytreating parents it is possible to correct many single gene disorderswhich otherwise might affect their children. It is similarly possible toalter the expression of fully inheritable disorders or those disordershaving at least a partially inherited basis, which are caused byinteraction of more than one gene, or those which are more prevalentbecause of the contribution of multiple genes. This technology can alsobe applied in a similar way to correct disorders in animals other thanhuman primates. In some instances, it may be necessary to introduce oneor more “gene(s)” into the germ cells of the animal to attain a desiredtherapeutic effect, as in the case where multiple genes are involved inthe expression or suppression of a defined trait. In the human, examplesof multigenic disorders include diabetes mellitus caused by deficientproduction of, or response to, insulin, inflammatory bowel disease,certain forms of atheromatous cardiovascular disease and hypertension,schizophrenia and some forms of chronic depressive disorders, amongothers. In some cases, one gene can encode an expressible product,whereas another gene encodes a regulatory function, as is known in theart. Other examples are those where homologous recombinant methods areapplied to repair point mutations or deletions in the genome,inactivation of a gene causing pathogenesis or disease, or insertion ofa gene that is expressed in a dominant negative manner, or alterationsof regulating elements such as gene promoters, enhancers, theuntranslated tail region of a gene, or regulation of expansion ofrepeated sequences of DNA which cause such diseases as Huntingdon'schorea, Fragile-X syndrome and the like.

A specific reproductive application of the present invention is to thetreatment of animals, particularly humans, with disorders ofspermatogenesis. Defective spermatogenesis or spermiogenesis frequentlyhas a genetic basis, that is, one or mutations in the genome can resultin failure of production of normal sperm cells. This can happen atvarious stages of the development of germ cells, and may result in maleinfertility or sterility. The present invention is applicable, forexample, to the insertion or incorporation of nucleic acid sequencesinto a recipient's genome and, thereby, establish sperrnatogenesis inthe correction of oligozoospermia or azoospermia in the treatment ofinfertility. Similarly, the present methods are also applicable to maleswhose subfertility or sterility is due to a motility disorder with agenetic basis.

The present invention is additionally applicable to the generation oftransgenic animals expressing agents which are of therapeutic benefitfor use in human and veterinary medicine or well being. Examples includethe production of pharmaceuticals in domestic cows' milk, such asfactors which enhance blood clotting for patients with types ofhaemophilia, or hormonal agents such as insulin and other peptidehormones.

The present method is further applicable to the generation of transgenicanimals, for example pigs, of a suitable anatomical and physiologicalphenotype for human xenograft transplantation. The inventive transgenictechnology permits the generation of animals which are immune-compatiblewith a human recipient. Appropriate organs, for example, can be removedfrom such animals to allow the transplantation of, for example, theheart, lung and kidney.

In addition, male germ cells genetically modified in accordance withthis invention can be obtained from the transgenic animal, and storedunder conditions effective for later use, as is known in the art.

The invention will now be described in greater detail by reference tothe following non-limiting examples. The pertinent portions of thecontents of all references, and published patent applications citedthroughout this patent necessary for enablement purposes are herebyincorporated by reference.

EXAMPLES Genetic Modification of Male Germ Cells In Vivo and In Vitro InVivo Adenovirus-enhanced Transferrin-polvlysine-mediated Delivery ofGreen Lantern Retorter Gene Delivery Svstem to Testicular Cells

The adenovirus enhanced transferrin-polylysine-mediated gene deliverysystem has been described and patented by Curiel et al. (Curiel D. T.,et al., Adenovirus enhancement of transferrin-polylysine-mediated genedelivery, PNAS USA 88: 8850-8854 (1991). The delivery ofDNA depends uponendocytosis mediated by the transferrin receptor (Wagner et al.,Transferrin-polycation conjugates as carriers for DNA uptake into cells,PNAS (USA) 87: 3410-3414 (1990). In addition this method relies on thecapacity of adenoviruses to disrupt cell vesicles, such as endosomes andrelease the contents entrapped therein. This system can enhance the genedelivery to mammalian cells by as much as 2,000 fold over other methods.

The gene delivery system employed for the in vivo experiments wasprepared as shown in examples below.

Example 1 Prevaration of Transferrin-poly-L-Lysine Complexes

Human transferrin was conjugated to poly (L-lysine) using EDC(1-ethyl-3-(3-dimethyl aminopropyl carbodiimide hydrochloride) (Pierce),according to the method of Gabarek and Gergely (Gabarek & Gergely,Zero-length cross-linking procedure with the use of active esters,Analyt. Biochem 185:131 (1990)). In this reaction, EDC reacts with acarboxyl group of human transferrin to form an amine-reactiveintermediate. The activated protein was allowed to react with the poly(L-lysine) moiety for 2 hrs at room temperature, and the reaction wasquenched by adding hydroxylamine to a final concentration of 10 mM. Theconjugate was purified by gel filtration, and stored at −20° C.

Example 2 Preparation of DNA for In Vivo Transfection

The Green Lantern-1 vector (Life Technologies, Gibco BRL, Gaithersberg,Md.) is a reporter construct used for monitoring gene transfection inmammalian cells. It consists of the gene encoding the Green FluorescentProtein (GFP) driven by the cytomegalovirus (CMV) immediate earlypromoter. Downstream of the gene is a SV40 polyadenylation signal. Cellstransfected with Green Lantern-1 fluoresce with a bright green lightwhen illuminated with blue light. The excitation peak is 490 nm.

Example 3 Preparation of Adenoviral Particles

Adenovirus dI312, a replication-incompetent strain deleted in the Elaregion, was propagated in the Ela trans-complementing cell line 293 asdescribed by Jones and Shenk (Jones and Shenk, PNAS USA (1979) 79:3665-3669). A large scale preparation of the virus was made using themethod of Mittereder and Trapnell (Mittereder et al., “Evaluation of theconcentration and bioactivity of adenovirus vectors for gene therapy”,J. Urology, 70: 7498-7509 (1996)). The virion concentration wasdetermined by UV spectroscopy, 1 absorbance unit being equivalent to 10viral particles/ml. The purified virus was stored at −70° C.

Example 4 Formation of Transferrin-poly-L Lysine-DNA-Viral Complexes

Six (6) micrograms of transferrin-polylysine complex from Example 1 weremixed in 7.3×10⁷ adenovirus d1312 particles prepared as in Example 3,and then mixed with 5 μg of the Green Lantern DNA construct of Example2, and allowed to stand at room temperature for 1 hour. About 100 μl ofthe mixture were drawn up into a micropipette, drawn on a pipettepuller, and slightly bent on a microforge. The filled micropipette wasthen attached to a picopump (Eppendorf), and the DNA complexes weredelivered under continuous pressure, in vivo to mice as described inExample 6.

Controls were run following the same procedure, but omitting thetransferrin-poly-lysine-DNA-viral complexes from the administeredmixture.

Example 5 Comparison of Adenovirus-enhanced Transferrin-polylysine &Lipofectin Mediated Transfection Efficiency

The conjugated adenovirus particle complexed with DNA were tested on CHOcells in vitro prior to in vivo testing. For these experiments aluciferase reporter gene was used due to the ease of quantifyingluciferase activity. The expression construct consists of a reportergene encoding luciferase, is driven by the CMV promoter (Invitrogen,Carlsbad, Calif. 92008). CHO cells were grown in Dulbecco's modifiedEagle's medium (DMEM) with 10% fetal calf serum. For gene transferexperiments CHO cells were seeded into 6 cm tissue culture plates andgrown to about 50% confluency (5×10⁵ cells). Prior to transfection themedium was aspirated and replaced with serum free DMEM. Cells wereeither transfected with transferrin-polylysine-DNA complexes or withlipofectin DNA aggregates. For the transferrin-polylysine mediated DNAtransfer, the DNA-adenovirus complexes were added to the cells at aconcentration of 0.05-3.2×10⁴ adenovirus particles per cell. Plates werereturned to the 5% CO₂ incubator for 1 hour at 37° C. After 1 hour 3 mlof complete media was added to the wells and the cells were allowed toincubate for 48 hours before harvesting. The cells were removed from theplate, counted and then lysed for measurement of luciferase activity.

For cells transfected by lipofectin, 1 μg of CMV-luciferase DNA wasincubated with 17 μl of Lipofectin (Life Technologies). TheDNA-lipofectin aggregates were added to the CHO cells and allowed toincubate at 37° C. at 5% CO₂ for 4 hours. Three milliliters of completemedium was added then to the cells and they were allowed to incubate for48 hours. The cells were harvested, counted and lysed for luciferaseactivity. The luciferase activity was measured by a luminometer. Theresults obtained are shown in Table 1.

The data included in Table 1 below show that the adenovirus-enhancedtransferrin-polylysine gene delivery system is 1,808 fold more efficientthan lipofection for transfection of CHO cells.

TABLE 1 Comparison of Lipofection & Adenovirus EnhancedTransferrin-polylysine Transfection of CHO Cells Luciferase ActivitySample Treatment (RLU) 1 1 × 10⁷ particles + 6 ug CMV-Luc 486 2 2.5 ×10⁷ particles + 6 ug CMV-Luc 1201 3 5.0 × 10⁷ particles + 6 ug CMV-luc11119 4 1 × 10⁹ particles + 6 ug CMV-Luc 2003503 5 Lipofection 1108 6Unmanipulated cells 155

Example 6 In Vivo Delivery of DNA to Animal's Germ Cells viaTransferrin-L-lysine-DNA-Viral Complexes

The GFP DNA-transferrin-polylysine viral complexes, prepared asdescribed in Example 4 above, were delivered into the seminiferoustubules of three (3)-week-old B6D2F1 male mice. The DNA delivery bytransferrin receptor-mediated endocytosis is described by Schmidt et al.and Wagner et al. (Schmidt et al., Cell 4: 41-51 (1986); Wagner, E., etal. PNAS (1990), (USA) 81: 3410-3414 [1990]). In addition, this deliverysystem relies on the capacity of adenoviruses to disrupt cell vesicles,such as endosomes and release the contents entrapped therein. Thetransfection efficiency ofthis system is almost 2,000 fold higher thanlipofection.

The male mice were anesthetized with 2% Avertin (100% Avertin comprises10 g 2,2,2-tribromoethanol (Aldrich) and 10 ml t-amyl alcohol (Sigma),and a sriiall incision made in their skin and body wall, on the ventralside of the body at the level of the hind leg. The animal's testis waspulled out through the opening by grasping at the testis fat pad withforceps, and the vas efferens tubules exposed and supported by a glasssyringe. The GFP DNA-transferrin-polylysine viral complexes wereinjected into a single vasa efferentia using a glass micropipetteattached to a hand held glass syringe or a pressurized automaticpipettor (Eppendorf), and Trypan blue added to visualize the entry ofthe mixture into the seminiferous tubules. The testes were then placedback in the body cavity, the body wall was sutured, the skin closed withwound clips, and the animal allowed to recover on a warm pad.

Example 7 Detection of DNA and Transcribed Message

Nine (9) days after delivery of the genetic material to the animals'testis, two of the animals were sacrificed, their testes removed, cut inhalf, and frozen in liquid nitrogen. The DNA from one half of thetissues, and the RNA from the other half of the tissues were extractedand analyzed.

(a) Detection of DNA

The presence of GFP DNA in the extracts was tested 9 days afteradministration of the transfection mixture using the polymerase chainreaction, and GFP specific oligonucleotides. GFP DNA was present in thetestes of the animals that had received the DNA complexes, but wasabsent from sham operated animals.

(b) Detection of RNA

The presence of GFP mRNA was assayed in the testes of experimentalanimals as follows. RNA was extracted from injected, and non-injectedtestes, and the presence of the GFP messages was detected using reversetranscriptase PCR (RT PCR) with GFP specific primers. The GFP messagewas present in the injected testes, but not in the control testes. TheDNA detected above by PCR analysis is episomal GFP DNA. The transfectedgene was being transiently expressed.

Example 8 Expression of Non-endogenous DNA

Two males, one having received an injection with the GFP transfectionmixture and a control to whom only surgery was administered, weresacrificed 4 days after injection, and their testes excised, and fixedin 4% paraformaldehyde for 18 hours at 4° C. The fixed testis was thenplaced in 30% sucrose in PBS with 2 mM MgCl₂ for 18 hours at 4° C.,embedded in OCT frozen on dry ice, and sectioned. When the testes ofboth animals were examined with a confocal microscope with fluorescentlight at a wavelength of 488 nM, bright fluorescence was detected in thetubules of the GFP-injected mice, but not in the testes of the controls.Many cells within the seminferous tubules of the GFP-injected mouseshowed bright fluorescence, which evidences that they were expressingFluorescent Green Protein.

Example 9 Generation of Offspring from Normal Matings

GFP transfected males were mated with normal females. The females wereallowed to complete gestation, and the pups to be born. The pups (F1offspring or progeny) were screened for the presence of the novelgenetic material(s).

Example 10 In Vitro Transfection of Testicular Cells

Cells were isolated from the testes of three 10-day-old mice. The testeswere decapsulated and the seminiferous tubules were teased apart andminced with sterile needles. The cells were incubated in enzyme mixturefor 20 minutes at 37° C. The enzyme mixture was made up of 10 mg bovineserum albumin (embryo tested), 50 mg bovine pancreatic trypsin type III,Clostridium collagenase type I, 1 mg bovine pancreatic DNAse type I in10 mls of modified HTF medium (Irvine Scientific, Irvine, Calif.). Theenzymes were obtained from Sigma Company (St. Louis, Mo. 63178). Afterdigestion, the cells were washed twice by centrifugation at 500×g withHTF medium and resuspended in 250 μl HTF medium. The cells were counted,and 0.5×10⁶ cells were plated in a 60 mm culture dish in a total volumeof 5 ml DMEM (Gibco-BRL, Life Technologies, Gaithesburg, Md. 20884). Atransfection mixture was prepared by mixing 5 μg Green Lantern DNA(Gibco-BRL, Life Technologies, Gaithesburg, Md. 20884) with 20 μlSuperfect (Qiagen, Santa Clarita, Calif. 91355) and 150 μl DMEM. Thetransfection mix was added to the cells and they were allowed toincubate for 3 hours at 37° C., 5% CO₂ The cells were transferred to a33° C. incubator and incubated overnight.

The following morning the cells were assessed for transfectionefficiency by counting the number of fluorescent cells. In thisexperiment the transfection efficiency was 90% (Figure not shown). Thetesticular cells transfected with Green Lantern viewed with Nomaskioptics ×20 show the same cells viewed with FITC. Nearly all the cellswere fluorescent, which is confirmation of their successfultransfection.

Example 11 Preparation of a Cell Suspension from Testicular Tissue forCryopreservation

A cell suspension was prepared from mice of different ages as describedbelow.

Group I:  7-10 day olds Group II: 15-17 day olds Group III: 24-26 dayolds

The mice's testes were dissected, placed in phosphate buffered saline(PBS) decapsulated, and the seminiferous tubules were teased apart.Seminiferous tubules from groups I and II were transferred to HEPESbuffered culture medium (D-MEM) (Gibco-BRL, Life Technologies,Gaithersberg, Md. 20884) containing 1 mg/ml Bovine serum albumin (BSA)(Sigma, St. Louis, Mo. 63178) and Collagenase Type I (Sigma) for theremoval of interstitial cells. After a 10 minute incubation at 33° C.,the tubules were lifted into fresh culture medium. This enzymaticdigestion was not carried out on the testes from group I because oftheir fragility.

The tubules from group II and III mice or the whole tissue from group Imice were transferred to a Petri dish with culture medium and were cutinto 0.1-1 mm pieces using a sterile scalpel and needle. The mincedtissue was centrifuged at 500×g for 5 minutes and the pellet wasresuspended in 1 ml of enzyme mix. The enzyme mix was made up in D-DMEMwith HEPES (Gibco-BRL) and consisted of 1 mg/ml bovine serum albumin(BSA) (Sigma, embryo tested), 1 mg/ml collagenase I (Sigma) and 5 mg/mlbovine pancreatic trypsin (Sigma) and 0.1 mg/ml deoxyribonuclease I(DN-EP, Sigma). The tubules were incubated in enzyme mix for 30 minutesat 33° C. After the incubation, 1 ml of medium was added to the mix andthe cells were centrifuged at 500×g for 5 min. The cells were washedtwice in medium by centrifugation and resuspension. After the final washthe cell pellet was resuspended in 250 μl of culture medium and counted.

Example 12 Transferring Transfected Male Germ Cells into RecipientTestis

The cells were injected into the testis via the vasa efferentia using amicropipette. 3×10⁵ cells in a total volume of 50 μl were used for theinjection. The cells were mixed with Trypan blue prior to the injection.The recipient mice were anesthetized with 0.017 mL/g body wt. Avertin.An incision was made across the lower abdominal wall and the testis wasgently pulled to the exterior through the incision by pulling on the fatpad associated with the testis. The vas efferens was exposed andapproximately 20 μL of cell suspension was injected into the vasefferens using a glass micropipette held in a steel micropipette holder(Leitz). The cells were expelled from the pipette using air pressurefrom a 20 mL glass syringe. Prior to the transfer oftransfected germcells to the recipient animals, the recipient testes were depopulated ofendogenous male germ cells.

Example 13 Depopulating the Recipient Testis of Male Germ CellsComparison of Depopulating Treatments

Eight-week-old C57BL/6J mice were allowed to acclimatize for a few daysand then were assigned to one of the following three treatment groups.They received: (1) 400 Rad gamma irradiation; (2) 4 μG/g body weight ofbusulfan (1,4-butanediol dimethanesulphonate; Myleran, Glaxo Wellcome);or (3) a combination treatment of busulfan (4μg/g body wt) followed oneweek later by 400 Rad of gamma irradiation (“busulfan/400 Rad”treatment). A fourth group of untreated C57BL/6J mice of the same age asthe treatment groups was used as a control. There were 24 mice in eachtreatment group, and 3 mice were mice sacrified at each of the followingtime intervals after treatment: 5 hours, 24 hours, 48 hours, 72 hours, 1week, 2 weeks, 1 month and 2 months after treatment.

In addition, other C57BL/6J mice receiving the combined busulfan/400 Radtreatment were examined histologically at time points up to five monthsafter treatment (the testes of these other mice were fixed overnight in4% paraformaldehyde in PBS, pH 7.4 at 4° C., dehydrated and embedded inparaffin before sectioning and H&E staining).

Delivery of an Alkylating Agent to Recipient Vertebrates. The male micereceiving busulfan received a dose of 4 μg busulfan per g body wt. Thebusulfan was first dissolved 8 mg/mL in 100% dimethyl sulfoxide (DMSO)then, immediately before injection, was diluted 1:1 in phosphatebuffered saline, pH 7.4. The mice were injected with the dilutedbusulfan solution intraperitoneally.

Irradiation Treatment of Recipient Vertebrates. For the gammairradiation tre atmeit, mice were anesthetized with 0.017 mL/g body wt.of 2.5% Avertin. Gamma irradiation was specifically directed to thetestis in the following manner. Each mouse was placed in a lead chamberwith only the testis and lower abdomen exposed through elliptical holesto the irradiating source (¹³⁷Cs Gammacell 40 irradiator [Nordion]).There were six aligned holes in the floor and roof of the chamberthrough which the gamma radiation passed unobstructed. After irradiationthe animals were allowed to recover from the anesthesia on a warmheating pad or water bed until they regained consciousness.

Histology. At selected time points, mice from each treatment group wereeuthanized, and testicular tissues to be examined were fixed in 10%formalin in PBS, pH 7.4, at 4° C. for 24 hours. Small slits in thetestis capsule were made to allow penetration of the fixative. Fixedsamples were washed four times with PBS, and embedded in paraffin usinga Tissue Tek-II tissue processor (MET). Sections of 8 μm thickness werecut, stained with haemotoxylin and eosin (H&E), and mounted withAquamount (Lerner Laboratories) on glass slides with coverslips. Thesections were viewed on a Zeiss or Olympic light microscope with a 40×objective lens (total magnification 400×).

Quantitative Histologic Analysis. Quantitative data were collected fromthe testes of two animals for each of the treatment groups at two monthsafter treatment. (Table 2). For the control group only one mouse wasused. The seminiferous tubules in a single section were counted using a5× objective on a Zeiss light microscope (50× total magnification).Individual seminiferous tubules were examined at 400× totalmagnification. Seminiferous tubules were considered severely damaged ifhardly any cells remained in the tubule, and the tubule consisted of abasement membrane with a single layer of cells, mostly spermatogonia,lying along the basement membrane. Moderately damaged tubules weretubules, in which some of the spermatogenic layers close to the lumenwere partially sloughed off. Spermatozoan heads were counted in thetubules and averaged over the total number of tubules counted.

Results of Histological Analysis. Obvious histological changes were notseen in the testis until two weeks after treatment. (Data no shown).After two weeks changes included severe disruption of spermatogenesis;all the mature spermatozoa were lost and no spermatids or spermatocyteswere present. A few Sertoli cell nuclei and spermatogonia weredetectable in the periphery along the basement membrane. Six weeks afterbusulfan/400 Rad treatment there was evidence of the re-establishment ofspermatogenesis. Some spermatids and spermatozoa were seen as well as afew spermatocytes.

By about 3 months most of the seminiferous tubules had at leastpartially recovered and all stages of spermatogenesis appear to berepresented. (Data not shown). Spermatogenesis had returned to normal atthis stage by five months after busulfan/400 Rad treatment.

The three treatment groups described above were also compared. The mostdramatic differences among the groups were seen at two months aftertreatment. At two months the mice that were treated with the combinedbusulfan/400 Rad gamma irradiation treatment showed the greatest numberof substantially depopulated seminiferous tubules. Seminiferous tubulesfrom this group also contained a smaller average number of sperm headsper seminiferous tubule and the greatest proportion of severely andmoderately damaged seminiferous tubules compared to the other treatmentgroups and the control mice. (Table 2). Treatment of the-mice witheither 400 Rad gamma irradiation or busulfan alone also resulted indamage to the spermatogenic process, including sloughing of cells intothe lumen of the tubule, and substantially fewer mature spermatozoanheads compared to the controls, but to a significantly lesser extentthan exemplified by the busulfan/400 Rad treatment group.

These results clearly demonstrate that a combination of treatment withan alkylating agent and gamma irradiation is a more effective method ofdepopulating a vertebrate testis of male germ cells than either of thetwo treatments alone.

TABLE 2 Comparison of Various Methods of Depopulating a VertebrateTestis of Male Germ Cells. No. No. No. Average No. Tubules SeverelyModerately Total No. sperm heads/ Treatment Counted damaged damagedSperm heads tubule Control 50 0 0 1733 in 50 tubules 35 Busulfan/400 R50 15 (30%) 3 (6%) 460 in 50 tubules 9 Busulfan/400R 57 14 (24%)  8(14%) 383 in 50 tubules 8 Busulfan 70 1 (1%) 5 (7%) 957 in 50 tubules 19Busulfan 69  3 (10%) 2 (3%) 764 in 50 tubules 15 400R 52 2 (4%) 1 (2%)1005 in 50 tubules 20 400R 41 2 (5%) 3 (7%) 827 in 41 tubules 20

Example 14 In vivo Transduction Using a Viral Vector

A retroviral vector was used to transduce (genetically alter or modify)male germ cells of mice in vivo. Specifically, a pseudo-typedHIV-derived viral vector (L. Naldini et al., In vivo gene delivery andstable transduction of nondividing cells by a Lentiviral vector, Science272:263-67 [1996]), was used, as modified by Carlos Lois to expressGreen Flourescent Protein (GFP) instead of the LacZ reporter gene, underthe transcriptional control of the CMV promoter (HR'GFP).

Recipient C57BL/6J mice were treated with busulfan 44 days prior toviral infection. C57BL/6J male mice were injected intraperitoneally with0.1 ml busulfan at a concentration of 1 mg/ml. The dose was 4 μgbusulfan/gm body wt. One pretreated mouse was anesthetized with Avertin(0.017 mls/gm body wt.), and a ventral midline incision was made and theright testis exposed.

The vas efferentia were dissected away from the fat, and ten microlitresof HIV-Ederived GFP vector, HR'GFP, at a titer of 1×10⁹ particles/mlwere injected into the seminiferous tubules of the right testis via thevas efferens of a busulfan-treated C57 BL/6J mouse. Injection was donewith a quartz glass micropipette attached to a Picospritzer II. ThePicospritzer was set at 80 psi and gave 1 second bursts upon manualdepression of a foot pedal. All the seminiferous tubules of the testiscan be reached with a single injection as the vas efferens leads to acommon chamber, the rete testis, from which all the tubules radiate. Theleft testis was not injected and was used as a control. Transduction ofthe testicular cells within the tubules was widespread.

Twenty one days after infection, the mouse was sacrificed and the testeswere fixed overnight in 4% paraformaldehyde in PBS, pH 7.4 at 4° C. Thetestes were washed three times in PBS and placed in 20% sucroseovernight at 4° C. The testes were frozen in OCT and sectioned at 8 μmon a cryostat. The sections were thawed to room temperature immersed inphosphate saline buffer and viewed on a Zeiss 310 confocal microscope.The laser was set at a wavelength of 488 nm.

Green fluorescence was seen in all the seminiferous tubules that wereviewed, although the intensity was greatest in the tubules at thesurface of the testis. Transduction was seen in the Sertoli supportcells as well as in the spermatogonia along the basement membrane, butlittle was seen in the spermatocytes or sperrnatids. Very few maturespermatozoa were present due to the Busulfan treatment. No fluorescencewas seen in the left testes used as control. This shows that male germcells can be transduced by a viral vector and that the transduced geneis expressed.

Example 15 Optimization Microinjection of Gene Deliverv Mixture throughthe Vas Efferens

The method of delivery (Winston, R. M. L., Microsurgical reanastomosisof the rabbit oviduct and its functional and pathological sequelae,Brit. J. Obstet. Gynaecol. 82:513-522 ([1975]) of viral particles into asingle vas efferens, and thence to the seminiferous tubules, was firstoptimized in several mice (FIGS. 1 a-1 d). The mice were treated withbusulfan (Myleran: Glaxo Wellcome) 14 days before microsurgery tomaximize the chance of viral particles gaining access to spermatogonia,which lie on the basement lamella of the tubules. Busulfan, analkylating cytotoxic agent, depopulates the testis (Bucci, L. andMeistrich, M., Effects of busulfan on murine spermatogenesis:cytotoxicity, sterility, sperm abnormalities, and dominant lethalmutations, Mut. Res. 176:259-268 [1987]). At the intraperitoneal (IP)dose given (4 μg/g body wt.) many of the spermatocytes, spermatids andspermatozoa were eliminated from the tubules, but the testis recoveredthree to four months afterward and fertility was restored. This impliesthat stem cells remain viable and can repopulate the testis. Stem cellspermatogonia are known to be resistant to insults, often surviving whenother germ cell types are destroyed (Huckins, C. & Oakberg, W. F.,Morphological and quantitative analysis of spermatogonia in mouse testesusing whole mounted seminiferous tubules. II. The irradiated testes,Anat. Rec. 192:529-42 [1978]).

Example 16 Production of Transgenic Progeny by In Vivo Transduction ofMale Germ Cells Followed by Natural Mating

Microsurgery. After depopulation of the testis as described in Example15, viral particles were delivered to the seminiferous tubules asfollows: Mice were anaesthetised with isofluorane (0.5-2% in oxygen).Each testis was exposed through a midline abdominal incision. Using amicrosurgical approach (Winston [1975]; Zeiss microscope atmagnification 4 to 50×) the tissue bundle containing the vasa efferentiawas visualised (FIG. 1 a-1 b). Dissection from the surrounding fat wasaided by a stream of phosphate buffered saline forced through a fineneedle. A quartz glass micropipette was back-filled with 10 μL viralparticles (10⁹ pfu/ml) mixed with 1 μL polybrene (80 mg/mL). This wasattached to a micropipette (Eppendorf) and the particles introduced intothe vas efferens under 2.2 bar pressure in pulses of 1.5 seconds,controlled by foot pedal. Earlier trials using 1% Bromophenol dye showedthat most seminiferous tubules could be filled (FIG. 1 c), but duringtreatments, no dye was used and small air bubbles were introduced intothe liquid containing viral particles to confirm dispersion into theseminiferous tubules (FIG. 1 d). To preserve fertility, only single vasaefferentia were injected, reducing injury to the remaining ducts.

Preparation of the Viral Vector. The plasmid, pHR′-CMVLacZ (L. Naldiniet al. [1996]), was modified by replacing the BamHI-XhoI fragmentcontaining the LacZ gene with a fragment containing the EGFP gene(‘humanised’ GFP, Clontech). For the production of viral particles 40 μgplasmid DNA was used to transfect a 10-cm plate of 293T cells. The 40 μgof plasmid DNA was made up of 10 μg pCMV R9, 20 μg of modified pHR′ and10 μg envelope plasmid. Vesicular-stomatitis-virus-glycoprotein (VSV-G)pseudotyped vectors were produced by contransfection of the vectorplasmid with the Moloney murine leukemia virus (MLV) gag-pol packagingplasmid pCMV-GAGPOL and the VSV-G plasmid. The supernatant was harvested48-60 hours after transfection, subjected to high speed centrifugation,filtered through 0.45 μm filters and assayed. The transducing viralparticles had the MLV restricted envelope protein, env, substituted witha broad-spectrum env protein from the vesicular stomatitis virus.

In Vivo Transduction of Male Germ Cells. Six mice were now treated withviral particles containing the transducing vector pHR′ (10⁹particles/mL). A single vas efferens was injected with a volume of 10 μLretroviral concentrate together with 1 μL (80 mg/mL) polybrene. After 24days the mice were sacrificed and the testes removed and fixed forcryosectioning and histological examination. Testes were fixed for 48hours in 4% Paraformaldehyde pH 7.4, and placed in 20% sucrose inphosphate saline buffer pH 7.4 at 4° C. for 24 hours. They were embeddedin OCT and stored at −70° C. They were cryosectioned at 8 μm and viewedin a Zeiss 410 confocal microscope (FIG. 2).

Nearly all tubules sectioned contained cells expressing GFP. Expressionwas highest in Sertoli and spermatogonia cells (FIG. 2 a-b).

Natural Matings with Females after Transduction of Male Germ Cells.Eleven C57/B1/6J young males were then selected to test whethertransduced male germ cells could transmit the retrovirally integratedtransgene to the next generation. Six of these mice were treated with abolus of busulfan (IP; 4 μg/gm body wt.) 14 days before in vivotransduction microsurgery in accordance with the in vivo method ofincorporating exogenous genetic material into the genome of avertebrate, as described above, and three received the same dose onlyone week before in vivo transduction. Two other mice were notpre-treated with busulfan before the in vivo transduction operation.Lentiviral particles were introduced into the seminiferous tubules.After 14 weeks, B6D2F1 females were introduced into cages with themales. Transduced males fathered at least two successive litters.Litters were conceived 14, 15, 19 and 20 weeks after transduction. Allthe males, except one dying immediately after surgery, fatheredtransgenic offspring. (Table 3).

TABLE 3 Production of transgenic offspring per litter fathered bytreated males at various times after mating. Mouse # Pre-treatment 14weeks 15 weeks 19 weeks 20 weeks 1 Busulfan 1 week — 2/9 (22%) 8/10(90%)  0/9 (0%)  2 Busulfan 1 week — 1/7 (14%) 1/7 (14%) 2/7 (28%) 3Busulfan 1 week 4/7 (57%) 1/8 (12%) 4/6 (66%) 0/7 (0%)  4 Busulfan 1week 7/8 (87%) 3/7 (43%) 1/6 (17%) 1/8 (12%) 5 Busulfan 2 weeks 5/6(83%) 8/9 (89%) — 0/8 (0%)  6 Busulfan 2 weeks — 2/8 (25%)  8/8 (100%)1/9 (11%)  7* Busulfan 2 weeks — — — — 8 Busulfan 2 weeks —  6/6 (100%)— 1/8 (12%) 9 Busulfan 2 weeks —  8/8 (100%) — 3/10 (30%)   10** none2/5 (40%) 5/6 (83%) — — 11  none 3/7 (43%) 7/8 (88%) — 0/6 (0%)  *MouseNo. 7 died immediately after surgery; **Mouse No. 10 died 17 weeks aftersurgery.

PCR and Southern blot analysis of DNA from embryonic offspring. Embryosat approximately embryonic day 12.5, were screened for presence of thetransgene by polymerase chain reaction (PCR) and Southern blot analysis.For the PCR, GFP specific primers were used and a radiolabeled GFP cDNAprobe was used for the Southern blot analysis (FIG. 3). DNA was purifiedfrom embryos using the Gentra purification system. The presence of thetransgene was ascertained using PCR amplification with the following GFPspecific primers:

(A) forward primer: (SEQ. ID. NO.:1) 5′-GGT GAG CAA GGG CGA GGA GCT-3′(B) reverse primer: (SEQ. ID. NO.:2) 5′-TCG GGC ATG GCG GAC TTG AAG A-3′

The PCR cycling conditions were: denaturing 94° C. for 1 minute,annealing at 60° C. for 1 minute and extension at 72° C. for 3 minutes.PCR ran for 35 cycles and yielded a specific GFP product 470 base pairsin length. Each cycle step can be reduced to one second—“one second PCR”to yield a distinct 470-bp PCR amplification product. Southern blotanalysis was also done on the same embryo DNA extracts. The DNA was cutwith BamHI-XhoI, run on a 0.8% agarose gel and blotted overnight in 20×SSC onto Hydrobond XL paper. The blots were hybridised overnight at 65°C. with a ³²P-radiolabeled BamHI-XhoI GFP fragment isolated from thepHR'plasmid. The blots were washed at 65° C. (30 minutes) each in 2× SSCwith 0.1% SDS, 1× SSC with 0.1% SDS, 0.1 X SSC with 0.1% SDS and exposedto X-ray film.

PCR and Southern analysis showed that a high percentage of transgenicoffspring were obtained in litters conceived within 15 weeks. Theresults are summarized in Table 3. By 20 weeks the percentage oftransgenic progeny had dropped in all of the treatment groups, implyingthat the self-renewing spermatogonia were not transduced, but rather apopulation ofdifferentiating spermatogonia. Once the daughtercells fromthis population had matured and left the testis they were not renewed(Huckins, C. & Oakberg, W. F. [1978]). In Table 3, the ratios are thenumber of transgenic offspring out of the total number of embryos in thelitter.

Although pre-treatment with busulfan enhanced the transduction ofspermatogonia, mice untreated with busulfan also generated transgenicoffspring. Male germ cells take 60 days to differentiate fromspermatogonia (Russell, L. D., et al. In: Histological andHistopathological evaluation of the testis, Cache River Press [1990]),undergo meiosis and form spermatozoa. Since conception was more than 60days after transduction, it is presumed that the transgenic offspringwere conceived from differentiated daughter cells of transducedspermatogonia. EGFP expression was driven by the CMV promoter and wasevident in the testicular cells of the founder males 24 days afterinfection. The animals that were infected did not appear to have toxicside effects (Verma, I. M. and Somia, N. [1997]) with the possibleexception of one dying 17 weeks after surgery.

The foregoing examples being illustrative but not an exhaustivedescription of the embodiments of the present invention, the followingclaims are presented.

1-60. (canceled)
 61. An in vivo method of incorporating exogenousgenetic material into the genome of a non-human mammal, said methodcomprising: (a) injecting into a vas efferens, or rete of a testis of amale non-human mammal a gene delivery mixture comprising a retroviralvector that comprises at least one polynucleotide encoding a desiredgene product and optionally a gene encoding a desired gene produce andoptionally a gene encoding a genetic selection marker, operativelylinked to a promoter sequence such that a transcriptional unit isformed, under conditions effective to reach within the testis a malegerm cell selected from the group consisting of spermatogonial stemcells, type B spermatogonial, primary spermatocytes, preleptotenespermatocytes, leptotene spermatocytes, zygotene spermatocytes,pachytene spermatocytes, secondary spermatocytes, spermatids, andspermatozoa; (b) allowing the polynucleotide encoding a desired geneproduct to be incorporated into the genome of the male germ cell, sothat a genetically modified male gamete is produced by the malenon-human mammal; and (c) breeding the male non-human mammal with afemale non-human mammal of its species, such that the geneticallymodified gamete is united with a female gamete and a transgenic progenyis produced thereby, that carries the polynucleotide encoding thedesired gene product in its somatic cells.
 62. The method of claim 61,wherein the retroviral vector is selected from the group consisting ofMoloney murine leukemia virus vectors and pseudotyped lentiviralvectors.
 63. The method of claim 61, wherein the retroviral vector is alentiviral vector.
 64. The method of claim 61, wherein the retroviralvector is a pseudotyped Moloney murine leukemia virus vector.
 65. Themethod of claim 61, wherein breeding is accomplished by natural matingof the male non-human mammal and female non-human mammal.
 66. The methodof claim 61, wherein the retroviral vector further comprises a geneencoding a genetic selection marker operatively linked to a male germcell-specific promoter.
 67. The method of claim 61, wherein breeding isaccomplished by artificial insemination of the female non-human mammalwith semen comprising the genetically modified male gamete.
 68. Themethod of claim 61, wherein breeding is accomplished by in vitrofertilization of an ovum of the female non-human mammal with thegenetically modified male gamete.
 69. The method of claim 61, whereinbreeding is accomplished by intracytoplasmic sperm injection, subzonalinsemination, or partial zona dissection, resulting in fertilization ofan ovum of the female non-human mammal with the genetically modifiedmale gamete.
 70. The method of claim 61, wherein the polynucleotideencoding a desired gene product is operatively linked to a constitutivepromoter.
 71. The method of claim 61, wherein the polynucleotideencoding a desired gene product is operatively linked to acytokine-inducible promoter.
 72. The method of claim 61, wherein thepolynucleotide encoding a desired gene product is operatively linked toa tissue-specific promoter.
 73. The method of claim 61, wherein thepolynucleotide encoding a desired gene product is operatively linked toa developmentally or temporally regulated promoter.
 74. The method ofclaim 61, wherein the polynucleotide comprises a gene encoding a geneticselection marker, and said gene is operatively linked to a constitutivepromoter.
 75. The method of claim 61, wherein the polynucleotideencoding a desired gene product is operatively linked to an exogenouslyinducible promoter.
 76. The method of claim 61, wherein thepolynucleotide comprises a gene encoding a genetic selection marker, andsaid gene is operatively linked to a tissue-specific promoter.
 77. Themethod of claim 61, wherein the polynucleotide comprises a gene encodinga genetic selection marker, and said gene is operatively linked to adevelopmentally or temporally regulated promoter.
 78. The method ofclaim 61, wherein the polynucleotide encoding the desired gene productencodes a human gene product.
 79. The method of claim 61, wherein thegenetic selection marker is a fluorescent protein or light-emittingprotein.
 80. The method of claim 61, wherein the gene delivery mixtureis injected into the vas efferens of the testis.
 81. The method of claim61, wherein the gene delivery mixture is injected into a seminferoustubule of the testis.